Immunohistochemical distinction between mesothelial and adenocarcinoma cells in serous effusions: a combination panel-based approach with a brief review of the literature.

BACKGROUND: The prognostic and therapeutic significance of differentiating adenocarcinoma (AC) from reactive mesothelium (RM) in effusions cannot be overemphasized. To avoid diagnostic errors, ancillary techniques like immunohistochemistry are employed. However, results vary and no universal standard has been accepted so far. OBJECTIVE: To study the combined diagnostic efficacy of epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), E-cadherin (EC), calretinin (CAL), desmin (DES) and vimentin (VIM) in distinguishing RM from AC cells in serous effusions. STUDY DESIGN: Unequivocally diagnosed cases of 39 adenocarcinomatous and 38 RM populations were studied using sections from 49 formalin-fixed, paraffin-embedded cell blocks. MATERIALS AND METHODS: The immunomarkers were applied on cell block sections using the avidin-biotin peroxidase technique. The distribution/intensity of immunostaining in mesothelial and AC cells were graded semiquantitatively. STATISTICAL ANALYSIS USED: Fischer's exact test was used to calculate the efficacy of individual markers and their combinations. RESULTS: EMA was the best single marker for AC, with 100% sensitivity and 97.37% specificity. For the mesothelial cells, CAL exhibited 100% sensitivity and 92.31% specificity. DES was more specific than CAL but had a poor sensitivity of 55.26%. EC, CEA and VIM had unsatisfactory predictive values precluding their use as individual diagnostic markers. Among the combinations, two panels--EMA+ AND (CAL- OR DES-) for ACs and CAL+ AND (EMA- OR CEA-) for RM had 100% specificities and sensitivities. CONCLUSIONS: Most panel studies on fluid cytology are based on the arbitrary use of individual markers with the best statistical values, leading to a less than accurate diagnostic assessment. We believe that statistical parameters calculated in combination provide for a more practical and objective evaluation as well as allowing for meaningful comparative studies.

Comparison between manual and automated total nucleated cell counts using the ADVIA 120 for pleural and peritoneal fluid samples from dogs, cats, horses, and alpacas.

BACKGROUND: Analysis of body fluids includes an estimate of total nucleated cell count (TNCC). Automated methods may enhance the accuracy and timeliness of TNCC results. OBJECTIVE: The purpose of this report was to assess the ability of the ADVIA 120 hematology analyzer to accurately count nucleated cells in pleural and peritoneal fluids from animals, compared with manual counts. METHODS: Pleural and peritoneal fluids submitted in EDTA tubes to our laboratory over a 17-month period were used in the study. TNCC/microL was determined by a manual method, using a hemocytometer, and by an automated method, using the ADVIA 120. Correlation of results was determined by Passing-Bablok regression, Bland-Altman plots, and Pearson correlation analysis. RESULTS: Samples from dogs (n=36), cats (n=36), horses (n=59), and alpacas (n=11) were analyzed. High correlation in TNCC between methods was found for peritoneal fluid (n=93, r=.959), pleural fluid (n=49, r=.966), and all fluids combined (n=142, r=.960) (P<.001). Variation between methods was greater in samples with TNCCs<1000/microL (r=.62, P<.001). The ADVIA systematically overestimated the number of cells in all fluid samples by 95 cells/microL (confidence interval=19.2-190.5/muL). CONCLUSION: The ADVIA 120 reliably determines TNCC in pleural and peritoneal effusions and can be recommended for routine veterinary laboratory analysis.

Analytical validation of the Sysmex XT-2000iV for cell counts in canine and feline effusions and concordance with cytologic diagnosis.

BACKGROUND: The Sysmex XT-2000iV is a hematology analyzer that combines laser and impedance technology. Its usefulness for determining cell counts in canine and feline intracavitary effusions has not yet been studied. OBJECTIVES: The objectives of this study were to evaluate the analytical performance of the Sysmex XT-2000iV for cell counts in effusions from dogs and cats, and to assess correlation with an impedance counter and concordance with diagnoses based on cytologic findings. METHODS: Effusions (43 pleural, 23 peritoneal, 6 pericardial) were analyzed from 32 dogs and 34 cats. Total nucleated cell count (TNCC), HCT, and RBC count were determined on the Sysmex and compared with those obtained on an impedance counter (Hemat 8, SEAC). Imprecision, linearity, and limit of detection were determined for the Sysmex. An algorithm was designed using quantitative and qualitative data from the Sysmex to classify the effusions and the results were compared with diagnoses based on cytologic findings. RESULTS: Intra-assay and interassay coefficients of variation on the Sysmex were variable. Linearity of TNCC was >or=0.993 for dogs and cats, with the exception of effusions from cats with feline infectious peritonitis, which had delta (Delta) TNC values >3.0. In comparison with the Hemat 8, a proportional error was found for TNCC on the Sysmex. Effusion classification based on the algorithm was concordant with that obtained by cytologic examination in 43/72 (60%) samples. Discordant results usually were due to the misclassification of cells with similar morphology (such as mesothelial and carcinoma cells) in Sysmex scattergrams. CONCLUSION: The Sysmex XT-2000iV provides a precise and accurate TNCC and has moderate concordance with cytologic findings for classifying canine and feline effusions. Although microscopic examination of effusions is necessary to achieve an accurate diagnosis, the Sysmex can provide preliminary information that may be helpful to cytopathologists.

A new electrocautery pleural biopsy technique using an insulated-tip diathermic knife during semirigid pleuroscopy.

BACKGROUND: The biopsy size obtained with standard flexible forceps (SFF) during semirigid pleuroscopy is often insufficient for pathological examination. An insulated-tip diathermic knife (IT knife) allows safe resection of a larger lesion during gastrointestinal endoscopy. We sought to validate an electrocautery pleural biopsy technique using the IT knife during semirigid pleuroscopy. We compared the diagnosis of specimens obtained using the IT knife and SFF in 20 subjects with unexplained pleural effusion, and reviewed pleuroscopic parameters such as complications, procedure time, and diameter of the specimens. METHODS: After injecting saline with lidocaine and epinephrine below the affected pleura, the lesion was incised in a circular shape with full thickness by manipulating the IT knife. RESULTS: Diagnostic yields from specimens obtained with the IT knife and SFF were 85% (17 of 20 cases) and 60% (12 of 20 cases), respectively. The IT knife biopsy was superior to SFF in 8 of 20 patients (malignant pleural mesothelioma in three, nonspecific inflammation in two, metastatic breast cancer in one, and tuberculosis in one). These pleural lesions revealed thickened, smooth abnormal appearances. The overall diagnostic yield for both IT knife and SFF was 100%. Median time of the procedure, from first pleural injection to specimen removal, was 21 min (range 12-92 min), and median diameter of specimen was 13 mm (range 6-23 mm). There were no severe complications during the procedure. CONCLUSIONS: Electrocautery biopsy using the IT knife during semirigid pleuroscopy has great potential for diagnosing smooth abnormal pleura which are difficult to biopsy with SFF.

[Influence of pleural fluid red blood cell count on the misidentification of transudates]. / Influencia del recuento de hematíes del líquido pleural en la identificación errónea de los trasudados.

BACKGROUND AND OBJECTIVE: Light's criteria misclassify a quarter of transudates as exudates. We assessed the influence of red blood cell counts on pleural lactate dehydrogenase (LDH) levels and, thereby, on the specificity of Light's criteria. PATIENTS AND METHOD: We retrospectively reviewed 1,312 consecutive patients with pleural effusion, of whom 1,014 were exudates and 298 transudates according to clinical criteria. The relationship between pleural erythrocytes and LDH using simple linear regression analysis, as well as the operating characteristics of Light's criteria, were assessed. Finally, a formula to correct pleural LDH levels, according to the erythrocyte count, was generated. RESULTS: There was a linear relationship between the pleural erythrocyte count and LDH levels (r = 0.44; p < 0.001). Light's criteria yielded 81% specificity in patients with pleural erythrocyte counts < or = 10.000 3 10(6)/l, as compared to 61% in a group with a higher erythrocyte counts (p < 0.01). The application of the LDH formula enabled the correct reclassification of 24 of 64 (37%) false exudates. CONCLUSIONS: A high pleural erythrocyte count, through its influence on the LDH levels, may lead to a transudate being misclassified as an exudate after applying Light's criteria.

What is your diagnosis? Pleural fluid from a neonatal Thoroughbred filly with pneumonia.

A 3-day-old filly was presented to the Cornell University Hospital for Animals with an umbilical hematoma and mild aspiration pneumonia. The foal underwent abdominal surgery for resection of the hematoma. Recovery was uneventful, but 3 days after surgery, the foal became progressively tachypneic. Imaging studies revealed bilateral pleural effusion and pleuropneumonia. Cytologic evaluation and bacterial culture of the pleural fluid from both sides of the chest revealed sterile exudates, consisting mostly of neutrophils, with fewer macrophages and lymphocytes. Pleural fluid macrophages contained variable amounts of purple-magenta globular material in their cytoplasm. A lighter colored granular precipitate was also seen throughout the background of the smears. Similar material was identified in a macrophage in a peripheral blood smear prepared 2 days after abdominal surgery. Large amounts of extracellular pink precipitate were also seen in the blood smear and persisted in the blood for 7 days after surgery. A protective lubricant, carboxymethylcellulose, had been instilled into the abdominal cavity during surgery to prevent intra-abdominal adhesions. The intracytoplasmic pigment within pleural fluid and blood macrophages and the extracellular precipitate in peripheral blood and pleural fluid smears was compatible with carboxymethylcellulose. The material was probably derived hematogenously and was considered an incidental finding. The pleuritis was attributed to exacerbation of the original aspiration pneumonia by the general anesthesia.

Rentabilidad del estudio citológico del líquido pleural en el derrame maligno / Accuracy of pleural fluid cytology in malignant effusions

Objetivo: Evaluar la rentabilidad de los estudios citológicos sucesivos del líquido pleural (LP) para diagnosticar malignidad y analizar la influencia que sobre aquella tienen el tiempo transcurrido entre los análisis,el tamaño del derrame y las características bioquímicas del LP. Métodos: Se revisaron retrospectivamente 1.427 pacientes con derrame pleural (DP), de los que 466 eran de causa maligna. En este último grupo se analizaron las citologías sucesivas, el tiempo transcurrido entre las mismas, las características bioquímicas del LP y el tamaño del DP. Resultados: La sensibilidad de una primera citología fue del 48,5%. Cuando un primer estudio citológico era negativo, un segundo era diagnóstico en el 28,6% de los casos, mientras que con dos citologías negativas un tercer estudio conseguía un 10,3% de positividades adicionales. El tipo de tumor condiciona la rentabilidad de la citología (66,5% en adenocarcinomas frente a 30,8% en mesoteliomas), pero no así el tiempo transcurrido entre los análisis citológicos sucesivos ni el tamaño del DP. De los parámetros bioquímicos del LP, un análisis multivariante mostró que sólo un cociente entre la glucosa del LP y del suero ≤ 0,75 se relacionaba con una mayor sensibilidad de la citología (74 vs. 47%, p < 0,001). Conclusión: Se aconseja repetir al menos una segunda citología en todo DP de etiología incierta, cuando una primera ha resultado negativa. Este segundo estudio se puede realizar de forma inmediata ya que el paso del tiempo no incrementa la rentabilidad. El porcentaje de positividades está influido por el tipo de tumor y por algunas características bioquímicas del LP, como el cociente entre la glucosa del LP y del suero

Objective: To assess the usefulness of repeat cytological examination of pleural fluid (PF) for diagnosing malignancy as well as the influence of time length between analyses, effusion’s size and pleural fluid biochemistries on the diagnostic yield of cytology. Methods: Retrospective analysis of 1,427 patients with pleural effusion (PE), including 466 patients with malignant PE. In this latter group, the time length between cytological analysis, the size of the PE, and the biochemical characteristics of PF were recorded. Results: The first cytological analysis had a sensitivity of 48.5%. Ifthis was negative, a second PF specimen was diagnostic in 28.6% of cases, whereas submission of a third PF specimen allowed 10.3% of additional diagnosis. The incidence of positive results depended on the primary tumor (e.g. 66.5% in adenocarcinomas, 30.8% in mesotheliomas), but neither on the time length between cytological analyses nor on the effusion’s size. A multivariate analysis showed that a PF to serum glucoseratio ≤ 0.75 was associated with a higher diagnostic yield of cytology (74 vs. 47%, p < 0.001). Conclusion: At least a second PF specimen should be submitted immediately for cytologic analyis in all PE of unknown cause, when the first analysis is not contributory. To delay this second analysis does not increase diagnostic yield. The percentage of cases in which cytologic study of the PF established the diagnosis of malignant PE depends on the tumor type and on certain PF biochemical characteristics such as the PF to serum glucose ratio

TLR2 and TLR4 expression on the immune cells of tuberculous pleural fluid.

Toll-like receptors (TLRs) play an important role in mediating the down stream signaling of immune response in tuberculosis. The predominance of Th1 response in tuberculous pleurisy prompted us to study the expression profiles of TLR2 and TLR4 on different immune cells and on subsets of T cells obtained from the site of infection. Our results showed that TLR2 was up-regulated on the monocytes from pleural fluid indicating a prominent role for this receptor in anti-tuberculous immunity. Notably, TLR2 and TLR4 expression were also enhanced on IFN-gamma secreting CD4(+)T cells. However, their expression was down-regulated on activated and IL-4 secreting CD4(+)T cells from the site of infection indicating that TLR expression is differentially modulated on the different subsets of T cells depending on their activation status and cytokine expression. The down-regulation of both TLRs on the natural regulatory T cells despite their higher number at the site of infection might be a mechanism to maintain their suppressive activity.

The value of pleural fluid analysis.

Pleural fluid analysis in isolation may have clinical value. To have the greatest diagnostic impact, the clinician must formulate a prethoracentesis diagnosis based on the clinical presentation, blood tests, and radiographic imaging. With this approach, a definitive or confident clinical diagnosis can be expected in up to 95% of patients. The information in this report should allow the clinician to achieve this goal.

Diagnostic work-up of pleural effusions.

A wide range of diseases may be the cause of an accumulation of fluid in the pleural space. Pleural effusion is a major diagnostic problem, since the pleura is an inner cavity with no direct access. The aim of this review is to provide a practical approach to the investigation of the patient presenting with pleural effusion. This should help to accurately diagnose pleural effusion and keep time-consuming, but necessary, invasive investigations to the minimum.

Peripheral CD4+ T cells showing a Th2 phenotype in a patient with Mikulicz's disease associated with lymphadenopathy and pleural effusion.

Mikulicz's disease (MD) is a unique IgG4-related systemic disease indicated by enlargement of the lachrymal and salivary glands and which differs substantially from Sjögren's syndrome. A male patient with pleural effusion, swelling of the submandibular glands, and swelling of the paraaortic, mediastinal, and pararenal lymph nodes was diagnosed with MD. Analysis of peripheral CD4+ T cells from the patient revealed deviation of the Th1/Th2 balance to Th2. Prednisolone therapy ameliorated the disease and corrected the Th1/Th2 imbalance.

[Tuberculous pleurisy after tumour necrosis factor-alpha antagonist usage: case report]. / Tümör nekroz faktörü-alpha antagonisti kullanimi sonrasi gelisen tüberküloz plörezi: Olgu sunumu.

A thirty-six year old male patient presented with dyspnea, right-sided chest pain, night sweats and intermittent fever. He has a history of ankylosing spondylitis treated with tumour necrosis factor-alpha (TNF-alpha) antagonist (infliximab). Computed tomography of the chest showed mediastinal lymphadenopathy, right-sided pleural effusion, and atelectasis. The pleural fluid was exudative with lymphocyte dominance. Closed pleural biopsy was nondiagnostic. The adenosine deaminase level of the pleural fluid was 110 U/L. In light of these findings, the patient was diagnosed as tuberculous pleurisy and antituberculous treatment was given. After one month, pleural fluid was markedly reduced.

Herbal mixtures consisting of puerarin and either polyenylphosphatidylcholine or curcumin provide comprehensive protection against alcohol-related disorders in P rats receiving free choice water and 15% ethanol in pure water.

Chronic alcohol drinking has been associated with the development of a number of abnormalities, including neuron-behavioral disorders, liver, pancreas, and heart-related diseases and inflammation and immune disorders. Because diverse mechanisms are involved in the development of these disorders, the commonly used receptor- or enzyme-specific drugs do not provide comprehensive protection against the adverse effects of alcoholism. This study describes possible therapeutic potency of puerarin (PU) from kudzu root, polyenylphosphatidylcholine from soy (SPCh), and curcumin (CU) from turmeric against alcohol's addiction-related and inflammatory-related abnormalities in alcohol-preferring P rats receiving free choice water and 15% ethanol in water. P-rats were fed once daily either the vehicle (for control) or different doses of PU, SPCh, CU, PU + SPCh, or PU + CU. The rats were divided in two groups: one received water alone, and the other free choice water and ethanol. Four rats from each group were fitted with electroencephalogram (EEG) electrodes for EEG recording. After 70 days of alcohol drinking, alcohol was withdrawn for 2 weeks, and the withdrawal symptoms were assessed. This study showed that alcohol drinking for 70 days (1) caused liver inflammation characterized by elevated tumor necrosis factor-alpha, interleukin-1beta, and matrix metalloproteinase-9 expression and (2) dysregulated lipopolysaccharide (LPS)-induced pleurisy. Alcohol withdrawal after 70 days of drinking generated severe withdrawal symptoms including seizure-type EEG activity. PU suppressed the addiction-mediated abnormalities but did not affect the inflammation-related abnormalities, while SPCh or CU suppressed only the inflammation-related abnormalities in alcohol-drinking rats subjected to LPS-induced pleurisy. A combination of PU with SPCh or CU suppressed both the addiction-related and inflammation-related abnormalities of alcohol drinking. Therefore, a mixture consisting of PU and either SPCh or CU may provide alternative therapy for alcohol-related disorders.

Establishment of novel human dedifferentiated chondrosarcoma cell line with osteoblastic differentiation.

Dedifferentiated chondrosarcoma is a rare, highly malignant variant of chondrosarcoma in which a high-grade sarcoma coexists with a low-grade chondroid tumor. We herein review a case of dedifferentiated chondrosarcoma with an osteosarcoma omit component that occurred in the distal femur of a 38-year-old man. We established the cell line (NDCS-1) from a pleural effusion of the metastatic lung tumor. The cell line was characterized by a the G-banded karyotype, polymerase chain reaction (PCR) single-strand conformation polymorphism analysis, spectral karyotyping, and reverse transcriptase PCR (RT-PCR). The tumor exhibited complex karyotypes and a high frequency of chromosomal amplication with p53 mutation. This tumor revealed an osteoblastic and chondroblastic character in vitro and in severe combined immunodeficiency mice. The expression and phosphorylation of platelet-derived growth factor receptor-beta, which seemed to play a major role in the malignant phenotype of chondrosarcoma, was confirmed by RT-PCR and Western blotting. To our knowledge, this is the first report of the establishment of a human dedifferentiated chondrosarcoma.

Primary pleural epithelioid hemangioendothelioma with rhabdoid phenotype: report and review of the literature.

Epithelioid hemangioendothelioma (EHE) is a rare malignant vascular tumor described in diverse locations including lung and liver. Relative to these sites, primary EHE of the serous cavities is uncommon. EHE in the serous cavities mimics mesothelioma and adenocarcinoma clinically, radiographically, cytologically, and histologically. EHEs have plasmacytoid epithelioid cells with cytoplasmic vacuoles. In addition to these features, we noted eccentric nuclei with abundant eosinophilic cytoplasm and nuclei displaced peripherally by globular cytoplasmic inclusions imparting a "rhabdoid" phenotype. These cells were often seen surrounding a hyaline core. Rhabdoid features are not unique to a single entity, and a comprehensive immunohistochemical panel is essential. We report the occurrence of pleural EHE with rhabdoid features presenting in a pleural effusion, and review the literature of primary serosal EHEs.

CD4(+)CD25(+)FoxP3(+) regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease.

CD4(+)CD25(+) regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4(+) or CD8(+) T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4(+)CD25(+)FoxP3(+) regulatory T cells may modulate immunity against human tuberculosis (TB). Our results indicate that the number of CD4(+)CD25(+)FoxP3(+) Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4(+)CD25(+)FoxP3(+) Treg in pleural fluid inversely correlates with local MTB-specific immunity (p<0.002). These CD4(+)CD25(+)FoxP3(+) T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-gamma and IL-10 production in TB patients. Therefore, CD4(+)CD25(+)FoxP3(+) Treg expanded in TB patients suppress M. tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.

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.

Subarachnoid-pleural fistula as a complication of malignant pleural mesothelioma.

We report a 62-year-old male patient with asbestos-related malignant pleural mesothelioma who developed recurrent pleural effusions after surgical resection of paravertebral tumour masses. Pleural effusions were drained on several occasions with the patient suffering severe headaches and vascular dysregulation. Cytological studies of the pleural fluid showed no evidence of inflammatory or malignant cells. The fluid was interpreted as seroma despite its unusual transparency until magnetic resonance imaging was suggestive of a subarachnoid-pleural fistula; its presence was confirmed when beta-trace protein--a specific marker for cerebrospinal fluid--was added to the standard laboratory testing of the pleural effusion. A subarachnoid-pleural fistula has to be included in the differential diagnosis of patients with recurrent pleural effusions after surgical debulkment of malignant pleural mesothelioma. The beta-trace protein may help to establish this diagnosis especially in cases where important therapeutic consequences may need to be drawn.