Cytocentrifuge Slides Research Articles

Utilization of cytocentrifugation slides in flow cytometry laboratory: a tool for correlation of morphology and immunonophenotype

Cytocentrifuge slide (“cytospin”) is a simple and rapid technique that is mainly used in the cytology laboratory. We have routinely used the technique in flow cytometry (FCM) laboratory at Montefiore Medical Center for more than 15 years. Our aim is to illustrate the contribution of cytospin in direct cellular morphologic visualization in FCM immunophenotyping, in result interpretation, and additionally as a quality control tool. Four thousand two hundred forty-eight cases sent to our FCM Laboratory in the past 21 months were studied retrospectively. The clinical history, cytospins, immunophenotyping panels, FCM contour plots, final FCM reports, and pathology reports were reviewed. Morphologic examination of the cytospin is a cost-effective tool for sample quality screening. Of the 4248 immunophenotyping cases, 359 (8.4%) cases were canceled, according to laboratory protocol based on cytospin review. Morphologic analysis of the cytospin provides extra information that guides both selection of the FCM immunophenotype panel and gating the cells of interest. Additionally, morphologic analysis of cytospin can yield early diagnosis when cohesive benign or malignant non-hematologic cells are present or even infectious organisms such as parasites are identified. Finally, the cytospin is a convenient tool for morphology-phenotype correlation. Cytospin is cost-effective and provides important morphologic information that facilitates FCM immunophenotyping panel selection and result interpretation.

Validation of MIB-1 Proliferation Index Performed on Cytocentrifuge Slide Preparations

Validation of MIB-1 Proliferation Index Performed on Cytocentrifuge Slide Preparations

Protective effect of C4a against hyperoxic lung injury via a macrophage-dependent but not a neutrophil/lymphocyte-dependent signaling pathway.

Complement anaphylatoxins have been investigated extensively; however, the role of complement anaphylatoxin C4a in hyperoxic lung injury has yet to be investigated. To the best of our knowledge, the present study is the first to demonstrate the role of C4a in hyperoxic lung injury in vitro and in vivo. BALB/c mice were ventilated with 100% oxygen with or without C4a treatment for 36 h. The body weight and the relative lung weight of the mice were determined, along with any morphological changes in the lung. The expression levels of interleukin (IL)-1, IL-6 and tumor necrosis factor-α (TNF-α) were quantified in the lung tissue and bronchoalveolar lavage fluid (BALF) samples by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were determined using cytocentrifuge slides and a hemocytometer. Histamine release from total cells in the BALF was also analyzed. The relative mRNA expression levels of CD68, F4/80, CD64, CD19 and CD3 in the murine lung tissue were assessed by reverse transcription-quantitative polymerase chain reaction. The results revealed that hyperoxia induced lung injury and morphological changes, and increased the expression levels of IL-1, IL-6 and TNF-α, histamine release, the number of inflammatory cells, and the expression levels of CD68, F4/80, CD64, CD19 and CD3. The hyperoxia-induced morphological changes and inflammatory reaction were significantly attenuated in mice treated with C4a. Treatment with C4a also attenuated the increase in the total cell count, decreased the number of macrophages in the BALF, and suppressed the elevated mRNA expression levels of CD68 and F4/80 in the lung tissue samples. Conversely, treatment with C4a did not affect the number of neutrophils or lymphocytes in the BALF or the mRNA expression of CD64, CD19 and CD3 in lung tissue. In conclusion, C4a attenuated hyperoxic lung injury via a macrophage-dependent but not a neutrophil/lymphocyte-dependent pathway.

Targeting complement anaphylatoxin C5a receptor in hyperoxic lung injury in mice.

Receptor binding of complement anaphylatoxin C5a results in proinflammatory activation of numerous diseases, but the role of receptor-mediated action during hyperoxic lung injury has, to the best of our knowledge, not yet been investigated. The contribution of the C5a receptor (C5aR) to hyperoxic lung injury in mice was investigated in this study. The effect of C5aR on hyperoxic lung injury in Balb/c mice was examined employing a C5aR antagonist (C5aRA). The mice were ventilated with 100% oxygen for 36 h with or without the administration of C5aRA. C5aR expression levels in non-treated or 100% oxygen-treated mice were assessed by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. The body weight and the relative lung weight of the mice, and the morphological changes in the lung were then determined. The total cell counts and the number of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) were determined using cytocentrifuge slides and a hemocytometer. The levels of interleukin-6 (IL-6), monocyte chemotactic protein (MCP-1) and tumor necrosis factor-α (TNF-α) in BALF and the myeloperoxidase (MPO) activity in the lung tissue were measured by enzyme-linked immunosorbent assay. The relative levels of CD68 and F4/80 messenger ribonucleic acid (mRNA) expression in the lung tissue were detected by RT-PCR. The TNF-α, IL-6 and MCP-1 protein expression levels in the lung tissue were assessed by western blot analysis. The results revealed hyperoxia-induced morphological changes, lung injury and increased expression levels of C5aR in lung tissue. The hyperoxia-induced increases in the total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were all significantly reduced in the mice receiving C5aRA. Treatment with C5aRA also attenuated the morphological changes and reduced MPO activity, and CD68 and F4/80 mRNA expression levels in the lung tissue, as well as the levels of IL-6, MCP-1 and TNF-α in BALF and lung tissue. In conclusion, C5a-C5aR action accelerated hyperoxia-induced lung injury, but this hyperoxic lung injury was attenuated by treatment with C5aRA.

Examination of body fluids: preparation of slides and cell morphology.

1. Leilani Collins, MS MT(ASCP)SH CLS(NCA)[⇑][1] 1. is associate professor, Clinical Laboratory Science Program, University of Tennessee Health Sciences Center, Memphis TN 1. Address for correspondence: Leilani Collins MS MT(ASCP)SH CLS(NCA), associate professor, Clinical Laboratory Science Program, University of Tennessee Health Sciences Center, 930 Madison Avenue, Suite 670, Memphis TN 38163. (901) 448-6299. lcollins{at}utmem.edu. 1. Describe and calculate the proper dilution for preparation of a monolayer cytocentrifuge slide. 2. List cellular findings that may be present in any fluid. 3. Describe lining cells that may be found in CSF, serous, and synovial fluids. 4. Distinguish benign and malignant cells. 5. Compare distinguishing characteristics of significant crystals in synovial fluids. Specimens concentrated by centrifugation or cytocentrifugation are used to perform differential counts and assess cell morphology on body fluids. Using standard centrifugation, the specimen is centrifuged, the supernatant removed, and a slide made from the buffy coat if RBCs or the concentrated cells of the centrifugate are present.. In cytocentrifugation the specimen is transferred to a cytofunnel assembly. The specimen is centrifuged at 1000 RPM for 10 minutes allowing fluid to be absorbed into filter paper and cells to be concentrated in a small area of the slide. The slides prepared by either concentration method should be allowed to air dry and are then stained with Wright or Wright-Giemsa stain prior to examination. If it is not possible to prepare cell concentrations to assess morphology of nucleated cells and cells are differentiated while the cell count is performed, a lysing agent that enhances the nucleus of cells must be used to determine the category of the cells. In this article, preparation of slides refers to cytocentrifugation. To prepare a monolayer cytocentrifuge slide, which is optimal for nucleated cell identification, use the nucleated cell count (not the RBC count) to determine the saline dilution for the cytocentrifuge preparation. A consistent amount of undiluted or diluted fluid should be used to prepare a cytocentrifuge slide—usually 0.25 mL or five drops. A good monolayer preparation can be obtained if the nucleated cell count is less than 200/μL. If the nucleated cell count is greater than 200/μL, divide the cell count by 100 to… ABBREVIATIONS: ALL = acute lymphoblastic leukemia; AML = acute myeloblastic leukemia; CNS = central nervous system; CSF = cerebrospinal fluid; HIV = human immunodeficiency virus; RBC = red blood cell; WBC = white blood cell. 1. Describe and calculate the proper dilution for preparation of a monolayer cytocentrifuge slide. 2. List cellular findings that may be present in any fluid. 3. Describe lining cells that may be found in CSF, serous, and synovial fluids. 4. Distinguish benign and malignant cells. 5. Compare distinguishing characteristics of significant crystals in synovial fluids. [1]: #corresp-1

Miscellaneous findings in body fluids.

1. Leilani Collins, MS MT(ASCP)SH CLS(NCA)[⇑][1] 1. is associate professor, Clinical Laboratory Science Program, University of Tennessee Health Sciences Center, Memphis TN 1. Address for correspondence: Leilani Collins MS MT(ASCP)SH CLS(NCA), associate professor, Clinical Laboratory Science Program, University of Tennessee Health Sciences Center, 930 Madison Avenue, Suite 670, Memphis TN 38163. (901) 448-6299. lcollins{at}utmem.edu. 1. Describe the significance of eosinophils in urine. 2. Describe lupus erythematosus cells in serous and synovial fluids. 3. Describe the significant cellular findings in bronchial alveolar lavage (BAL) specimens. Cytocentrifuge slides can provide important information in diagnosing various conditions. Urine for eosinophils Urine samples for eosinophils may be submitted when interstitial nephritis secondary to antibiotic therapy is suspected. Slides to search for eosinophils should be prepared by concentration of the sample by either standard centrifugation or cytocentrifugation. Slides should be stained with Wright stain and a thorough search made for eosinophils. A finding of any eosinophils is significant. Lupus Erythematosus (LE) cells LE cells are neutrophils containing a smooth homogeneous nuclear mass. The nucleus of the neutrophil will be displaced to the edge of the cell wall by the large homogeneous mass. In patients who have systemic lupus erythematosus, the characteristic LE cell may be seen in serous and synovial fluids. All the factors necessary for formation of these cells—incubation, trauma to the cells, and the LE factor—are present in vivo in patients with systemic lupus erythematosus. The finding of these cells can be of diagnostic significance in patients not previously identified with this disease. See Figure 1. Bronchoalveolar lavage (BAL)—the fluid that isn't a fluid Bronchoalveolar lavage (BAL) is usually performed on hospitalized patients who have non-resolving pneumonia in an attempt to identify organisms that are not responding to antibiotic treatment or to detect malignancy. The “fluid” is obtained by introducing warmed saline into the lungs through a bronchoscope in 20 mL aliquots followed by aspiration of the saline. A total of 100-120 mL of saline is introduced and aspirated with a recovery of 40-60 mL.3 The “fluid”… ARDS = adult respiratory distress syndrome; BAL = bronchoalveolar lavage; LE = Lupus Erythematosus. 1. Describe the significance of eosinophils in urine. 2. Describe lupus erythematosus cells in serous and synovial fluids. 3. Describe the significant cellular findings in bronchial alveolar lavage (BAL) specimens. [1]: #corresp-1

Magnetic enrichment of bronchial epithelial cells from sputum for lung cancer diagnosis

Sputum is an easily accessible diagnostic material for lung cancer early detection by cytologic and molecular genetic analysis of exfoliated airway epithelial cells. However, the use of sputum is limited by its cellular heterogeneity, which includes >95% macrophages and neutrophils and only about 1% bronchial epithelial cells. We propose to obtain concentrated and purified bronchial epithelial cells to improve early detection of lung cancer in sputum samples. Sputum was collected from patients with stage I nonsmall-cell lung cancer, cancer-free smokers, and healthy nonsmokers. Magnetic-assisted cell sorting (MACS) with anti-CD14 and anti-CD16 antibody beads were used to enrich bronchial epithelial cells by depleting macrophages and neutrophils from sputum. Fluorescence in situ hybridization (FISH) analysis for detection of FHIT deletion and cytology were evaluated in the enriched specimens. The bronchial epithelial cells were concentrated to 40% purity from 1.1% of the starting population, yielding an average of 36-fold enrichment and at least 2.3 x 10(5) cells per sample. Detecting FHIT deletions for lung cancer diagnosis produced 58% sensitivity in the enriched sputum, whereas there was 42% sensitivity in the unenriched samples (P = .02). Cytologic examination of the enriched sputum resulted in 53% sensitivity, as compared with 39% sensitivity in unenriched sputum (P = .03). Furthermore, only 2 cytocentrifuge slides of the unenriched sputum were needed for the analyses, as compared with up to 10 cytocentrifuge slides required from the unprocessed specimens. The enrichment of bronchial epithelial cells could improve the diagnostic value of sputum and the efficiency of genetic and cytologic analysis of lung cancer.

AVALIAÇÃO CLÍNICA E CITOLÓGICA DO TRATO RESPIRATÓRIO DE CAVALOS DA RAÇA QUARTO DE MILHA, APÓS O EXERCÍCIO

O objetivo do presente estudo foi avaliar a citologia do lavado broncoalveolar (BAL) após o exercício de doze cavalos da raça Quarto de Milha, provenientes de Curitiba e Região Metropolitana. Após a infusão de 100 ml de solução fisiológica com auxílio de endoscopia e cateter de silicone, foram recuperados acondicionados em torno de 30 mL de BAL por cavalo, analisados e submetidos à centrifugação. Lâminas de citocentrífuga foram obtidas e coradas pelas técnicas de Giemsa, Papanicolaou e Azul da Prússia. Nenhum dos cavalos apresentou epistaxe ou outra anormalidade ao exame clínico, nem sangramento pulmonar à endoscopia. A contagem de hemácias e leucócitos no BAL esteve dentro dos valores de referência encontrados na literatura, assim como os diferenciais leucocitários, com a proporção média de 62,3±13,7% e 34,1±14,8% para macrófagos e linfócitos, respectivamente. No entanto, a presença de hemossiderófagos em diferentes percentuais, sugere que parte destes animais estejam sob hemorragia pulmonar incipiente, não detectada clinicamente. Concluiu-se, baseado no presente trabalho, que a avaliação das populações celulares com o uso da citocentrífuga em associação a colorações especiais pode fornecer ao clínico importantes informações adicionais acerca do status funcional do trato respiratório, em particular hemorragias subclínicas.

Cytometric analysis of BAL T cells labeled with a standardized antibody cocktail correlates with immunohistochemical staining

Determining T-cell phenotypes of lung cells obtained by bronchoalveolar lavage (BAL) is frequently clinically useful, particularly for evaluating causes of interstitial lung disease. The current standard of determining CD4/CD8 T-cell subsets by immunohistochemical (IHC) staining of cytocentrifuge slides is labor-intensive and subject to interpreter variation. Flow cytometry (FCM) is a precise and rapid method commonly used in research to characterize cells in the lung. However, few studies address the methodology of analysis of BAL lymphocytes by FCM. Patients underwent bronchoscopy for clinical purposes. A BAL cell differential and T-cell subtype was requested by the treating physician to supplement the evaluation of patients with suspected interstitial lung disease. We used a commercially available T-cell antibody reagent, approved for analysis of blood via FCM, for T-cell subtyping of clinical BAL specimens. The percentages of CD4 and CD8 T-cell populations, as well as the CD4/CD8 ratios showed excellent correlation with IHC staining of cytocentrifuge slides regardless of the acquisition program used, as long as the gating strategy remained consistent (r > or = 0.9693 for CD4, r > or = 0.9589 for CD8, and r > or = 0.9485 for the CD4/CD8 ratio). These findings validate the use of standardized, commercially available antibody cocktails for BAL lymphocyte subtyping, making this technique available to clinicians and researchers with access to a three-color or four-color flow cytometer.

Thyroid Transcription Factor–1 Immunocytochemical Staining of Pleural Fluid Cytocentrifuge Preparations for Detection of Small Cell Lung Carcinoma

To assess whether thyroid transcription factor-1 (TTF-1) immunostaining of cytocentrifuge slides previously stained by the Papanicolaou technique is feasible and useful in diagnosing small cell lung carcinoma (SCLC) in pleural fluid. Eleven Papanicoalou-stained cytocentrifuge preparations of pleural fluid from 11 patients with SCLC were reviewed. The initial cytologic diagnoses were 5 positive, 1 suspicious and 5 negativefor SCLC. After transferring the cytologic material to a positively charged slide, immunostaining was performed using mouse monoclonal antibody against TTF-1. The neoplastic cellularity varied among the single suspicious and 5 positive cases. The case with a suspicious diagnosis had scant tumor cells. All 6 cases showed nuclear stainingfor TTF-1 in most of the tumor cells. The other 5 cases with a negative cytologic diagnosis were negativefor TTF-1. The results of this study indicate that immunostaining for TTF-1 can be successfully applied to previously Pap-stained cytocentrifuge preparations to assist in the diagnosis of SCLC in pleural fluid.

Clinical Correlation of Asbestos Bodies in BAL Fluid

Asbestos bodies (AB) in BAL cells are specific markers of asbestos exposure. We retrospectively reviewed BAL cytocentrifuge slides of 30 utility workers with a history of asbestos exposure and 30 normal volunteers. BAL cytocentrifuge slides were blinded and scanned under 40 x light microscope. AB were found more frequently in subjects with a history of asbestos exposure compared to normal volunteers (10 of 30 subjects, 33%, vs 0 of 30 subjects). The mean number of AB seen in the AB-positive group was 2.7 per slide. Demographic data were comparable including age, gender, and smoking. Exposure histories were also similar: duration > 20 years, onset > 30 years ago, and time since last exposure > 7 years. More AB-positive patients reported respiratory symptoms (70% vs 26%, p < 0.05). High-resolution CT scans of AB-positive patients revealed a higher prevalence of parenchymal disease (70% vs 26%, p < 0.05). AB-positive subjects had reduced pulmonary function compared to AB-negative subjects: FVC (86% vs 97% predicted), FEV(1) (77% vs 92% predicted, p < 0.05), and diffusion capacity of the lung for carbon monoxide (76% vs 104% predicted, p < 0.01). In individuals with a history of asbestos exposure, the presence of AB in BAL cells is associated with higher prevalence of parenchymal abnormalities, respiratory symptoms, and reduced pulmonary function.

Alveolar Macrophage Graded Hemosiderin Score from Bronchoalveolar Lavage in Horses with Exercise‐Induced Pulmonary Hemorrhage and Controls

The objective of this study was to determine if a quantitative scoring system for evaluation of hemosiderin content of alveolar macrophages obtained by bronchoalevolar lavage provides a more sensitive test for the detection of exercise‐induced pulmonary hemorrhage (EIPH) in horses than does endoscopy of the lower airways. A sample population composed of 74 Standardbred racehorses aged 2–5 years was used. Horses were grouped as either control (EIPH‐negative) or EIPH‐positive based on history and repeated postexertional endoscopic evaluation of the bronchial airways. Bronchoalveolar lavage was performed and cytocen‐trifuge slides were stained with Perl's Prussian blue. Alveolar macrophages were scored for hemosiderin content by a method described by Golde and associates to obtain the total hemosiderin score (THS). Test performance criteria were determined with a contingency table. All subjects had some degree of hemosiderin in the alveolar macrophages, regardless of group. The distribution of cells among the different grades followed a significantly different pattern for the control group versus horses with EIPH (P &lt; .05). When using a THS of 75 as a cutoff point, the THS test was found to have a sensitivity of 94% and a specificity of 88%. The level of agreement beyond chance, between the EIPH status and the THS test result was very good (Cohen's kappa = 74%). The conclusion was made that careful assessment and scoring of alveolar macrophages for hemosiderin by means of the Golde scoring system shows promise as a more sensitive approach than repeated postexertional endoscopy alone to detect EIPH.

Charcot-Leyden Crystal Protein (Galectin-10) Is Not a Dual Function Galectin with Lysophospholipase Activity but Binds a Lysophospholipase Inhibitor in a Novel Structural Fashion

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys(29) and with Cys(57) near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp(72), in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.

Alveolar macrophage graded hemosiderin score from bronchoalveolar lavage in horses with exercise-induced pulmonary hemorrhage and controls.

The objective of this study was to determine if a quantitative scoring system for evaluation of hemosiderin content of alveolar macrophages obtained by bronchoalevolar lavage provides a more sensitive test for the detection of exercise-induced pulmonary hemorrhage (EIPH) in horses than does endoscopy of the lower airways. A sample population composed of 74 Standardbred racehorses aged 2-5 years was used. Horses were grouped as either control (EIPH-negative) or EIPH-positive based on history and repeated postexertional endoscopic evaluation of the bronchial airways. Bronchoalveolar lavage was performed and cytocentrifuge slides were stained with Perl's Prussian blue. Alveolar macrophages were scored for hemosiderin content by a method described by Golde and associates to obtain the total hemosiderin score (THS). Test performance criteria were determined with a contingency table. All subjects had some degree of hemosiderin in the alveolar macrophages, regardless of group. The distribution of cells among the different grades followed a significantly different pattern for the control group versus horses with EIPH (P < .05). When using a THS of 75 as a cutoff point, the THS test was found to have a sensitivity of 94% and a specificity of 88%. The level of agreement beyond chance, between the EIPH status and the THS test result was very good (Cohen's kappa = 74%). The conclusion was made that careful assessment and scoring of alveolar macrophages for hemosiderin by means of the Golde scoring system shows promise as a more sensitive approach than repeated postexertional endoscopy alone to detect EIPH.

IMMUNOSTAINING OF CYTOKERATIN 20 IN CELLS FROM VOIDED URINE FOR DETECTION OF BLADDER CANCER

Cytokeratin 20 is a cytoskeletal protein expressed in colon and bladder cancer cells but only rarely in normal urothelium. Previous studies have shown that identification of RNA coded for cytokeratin 20 in urine samples using polymerase chain reaction is highly sensitive for detection of bladder tumors. We examined the efficacy of immunocytology on cells from voided urine samples using monoclonal antibodies against cytokeratin 20 for noninvasive detection of bladder tumors. A total of 174 patients comprised the study, including 80 who were evaluated because of hematuria or irritative voiding symptoms and 94 who were examined during followup visits after resection of bladder tumors. Voided urine samples were obtained for immunocytology and cytopathology. Each patient underwent cystoscopy, and biopsies were done when a bladder tumor was detected or carcinoma in situ was suspected. Indirect immunoperoxidase staining was done on cytocentrifuge slides using a monoclonal antibody against cytokeratin 20. Bladder tumors were found in 87 patients. Immunocytology resulted in 81.6% sensitivity, 77% specificity and 80% accuracy. False-negative results occurred in 12 patients with superficial low grade tumors and in 4 with high grade invasive tumors. Cytopathology resulted in 51.7% sensitivity and 94.2% specificity. Immunocytology of the cytokeratin 20 antigen is significantly more sensitive than cytopathology for bladder tumor detection, especially low grade and low stage cancer. The results are not affected by inflammation or previous treatments with bacillus Calmette-Guerin. Immunocytology may miss high grade tumors and, therefore, cannot be used alone as a substitute for cystoscopy.

Immunocytochemical Study of Malignant Lymphoma in Serous Effusions

To cytologically evaluate a large series of serous effusions associated with malignant lymphoma (ML), identify the immunoreaction patterns of the cells from selected positive cases and to investigate the correlation of cytomorphology with tissue section diagnosis. From 1966 to 1990, a review of the files of the Department of Anatomic Pathology, A. C. Camargo Hospital, disclosed 4,297 cases of serous effusions, 256 of which were associated with ML. Cytopathologically positive cases were selected for immunocytochemical study. All paraffin sections stained with hematoxylin and eosin were reviewed to confirm the malignancy of the cases. Immunostaining was performed on both cytocentrifuge slides previously stained with Papanicolaou stain and new sections of the biopsy samples using the immunoperoxidase method and avidin-biotin complex with monoclonal mouse antihuman B-cell marker L-26 and monoclonal (mouse) antihuman T-cell marker UCHL-1. Immunocytochemical reactions were performed in 54 cases: 22 were pan-B positive and 10 pan-T positive; 24 cases showed no reactivity for either monoclonal antibody. Immunohistochemical reactions were performed in 24 available cases: 15 were pan-B positive and pan-T positive; 3 cases showed no reactivity for either monoclonal antibody. Cytohistoimmunoreactions were similar in 11 pan-B positive cases and 2 pan-T positive cases. Three cases were negative for both markers, 4 cases were pan-T positive in tissue samples and negative in cytocentrifuge smears, 3 cases were pan-B positive in tissue and negative on cytology and 3 cases were negative for both markers in both tissue and cytologic specimens. Cytology is an effective method of evaluating serous effusions associated with malignant lymphoma: no false positive diagnosis was observed in this series. Immunophenotyping of lymphoid cells is useful to classify and confirm the cytologic diagnosis.

Podocalyxin in Rat Platelets and Megakaryocytes

Podocalyxin is a membrane protein of rat podocytes and endothelial cells. It has not been described in other cell types, and no amino acid or DNA sequence data are available about it. Here we show that podocalyxin antigens are present in rat platelets and megakaryocytes. In resting platelets, the antigens are mainly intracellular but become surface exposed after thrombin stimulation, as shown by immunofluorescence and flow cytometry. By Western blotting, platelet podocalyxin has an apparent Mr of 140,000. Cytocentrifuge slides of rat bone marrow show that anti-podocalyxin antibodies recognize large polyploid cells also expressing CD62P, indicating that the cells are megakaryocytes. From a rat glomerular cDNA library we isolated a clone covering the carboxyl-terminal nucleotides of rat podocalyxin. Its putative transmembrane or intracellular domains are 100% or >93% identical, respectively, with the human and rabbit podocalyxin-like proteins. The truncated extracellular domain extends to include two of the four conserved cysteines shared by podocalyxin-like proteins. By Northern blotting, a 5.5-kb renal cortical transcript is seen. By in situ hybridization, cRNA probes recognize podocytes, endothelial cells, and megakaryocytes, and by reverse transcription polymerase chain reaction, platelets are shown to contain podocalyxin mRNA. Our results show that rat podocalyxin is a homologue of the previously cloned podocalyxin-like proteins and suggest that also in mammals podocalyxin has a role in hematopoiesis, as previously shown in the chicken.

Cell Block Preparation of a Burkitt’s Lymphoma Cell Line as a Positive Control for in Situ Hybridization for Epstein-Barr Virus

To examine the feasibility of the use of a cell block preparation of Epstein-Barr virus (EBV)-infected Burkitt's lymphoma cell line (EB-3) as a positive control for in situ hybridization (ISH) for EBV-encoded RNA (EBER). EB-3 cells were processed into cell block and cytocentrifuge preparations. ISH for EBER was performed, and the results were compared with a commercially available EBV positive control slide (cytocentrifuge). The cost of preparing the cell block and cytocentrifuge sample was only a fraction of the price of commercial control slides. ISH for EBER was strongly stained in all preparations with similar intensity of staining but with a much higher number of positive cells in the cell block. Although the cellular details in the cell block were considerably inferior to those on the cytocentrifuge and commercial control slide, with distortion of nuclei and smudging of chromatin, the interpretation of the ISH results was unaffected. The cell block was stable at room temperature when examined after one year. The cell block preparation of an EBV-infected Burkitt's lymphoma cell line serves as a reliable, stable and a relatively inexpensive control, with good preservation of cellular details of cellular RNA for ISH study for EBER in the detection of latent infection with EBV.

IMMUNOSTAINING OF LEWIS X IN CELLS FROM VOIDED URINE, CYTOPATHOLOGY AND ULTRASOUND FOR NONINVASIVE DETECTION OF BLADDER TUMORS

Purpose We examined the use of immunostaining of the Lewis X antigen in exfoliated cells from voided urine samples, cytopathology and bladder ultrasound for noninvasive detection of bladder tumors as a potential substitute for cystoscopy. Materials and Methods A total of 260 patients were included, of whom 80 were evaluated because of irritative symptoms or hematuria and 180 were examined during followup visits after resection of bladder tumors. Voided urine samples were obtained from each patient for immunocytology and cytopathology. Bladder ultrasound and cystoscopy were performed. Biopsies were obtained whenever a bladder tumor was seen or if carcinoma in situ was suspected. Indirect immunoperoxidase staining was done on cytocentrifuge slides, using the P12 monoclonal antibody against the Lewis X antigen. Results Cystoscopy and biopsies revealed bladder tumors in 84 patients. Immunocytology of 1 urine sample resulted in a sensitivity of 79.8% and a specificity of 86.4%. The diagnosis of primary carcinoma in situ by immunocytology was correct in 100% of the cases. The examination of 2 consecutive urine samples detected 95.1% of the tumors. False-negative results occurred in a few cases with small, superficial, low grade tumors. Cytopathology and bladder ultrasound resulted in a sensitivity of 47.6 and 66.7%, and a specificity of 97.7 and 97.2%, respectively. The results of immunocytology of 2 urine samples were equivalent to the combination of immunocytology of a single urine sample, cytology and ultrasound. Conclusions Immunostaining of the Lewis X antigen is significantly more sensitive than cytopathology for the detection of low grade as well as high grade tumor cells in voided urine. Immunocytological evaluation of 2 consecutive voided urine specimens for the Lewis X antigen is the most sensitive method currently available for noninvasive detection of transitional cell tumors. This assay may replace cystoscopy for detection of bladder cancer.

Wright-Giemsa Cytology of Body Fluids: Criteria for Identification of Malignant Cells

Cerebrospinal fluid and other body fluids from patients with known or suspected malignancy often are submitted to the laboratory It is therefore important to assess these samples carefully for the presence of malignant cells. Although Papanicolaou-stained preparations traditionally have been used for this purpose, Wright-Giemsa-stained cytocentrifuge slides are useful adjuncts in body fluid cytology and may improve the detection of leukemia and lymphoma. However, technologists and pathologists who examine Wright-Giemsa-stained cytocentrifuge slides must be familiar with the appearance of benign variants and malignant cells. This article presents criteria that are useful in making this distinction.