Use of narrow-range peptide IEF to improve detection of lung adenocarcinoma markers in plasma and pleural effusion.

In this study we applied narrow-range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow-range IEF were run; IPG strips and free-flow electrophoresis. Data from this study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n=3) were compared with inflammatory pleuritis (n=3) using iTRAQ quantification. Using narrow-range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue-related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies.

Perceptions vs. reality: measuring of pleural fluid pH in North Carolina.

BACKGROUND: Pleural fluid pH anaerobically handled and measured by a blood gas analyzer (BGA) is used to define a pleural space infection as complicated and predict the life expectancy of patients with malignant pleural effusions. Pleural fluid pH can also be measured by other less accurate methods. It is unknown whether physicians who use pleural fluid pH measurements are aware of the method used by their laboratories. METHODS: We surveyed 90 pulmonary physicians in North Carolina about their use of pleural fluid pH and their hospital laboratory's approach (pH indicator stick, pH meter, or BGA). We then contacted their hospital laboratories to determine the actual method of pH measurement. RESULTS: Twenty-eight (31%) pulmonologists in 11 North Carolina hospitals responded on their use of pleural fluid pH. Of the 20 pulmonologists who order pleural fluid pH, 90% reported that their hospital measures pleural fluid pH via BGA, but the majority (72%) were inaccurate. Only two of 11 hospitals reported that they measure pleural fluid pH with a BGA. CONCLUSION: Almost two-thirds of the chest physicians that order pleural fluid pH to help manage pleural effusions were using information that is not substantiated by the literature and, despite previous reports, hospitals still use suboptimal methods to measure pleural fluid pH. Further information is needed concerning the barriers to physicians and laboratory practices concerning the use of BGA for the measurement of pleural fluid pH.

Anthracotic pigment in pleural fluid: a case report.

BACKGROUND: The presence of anthracotic pigment (carbon) in pleural fluid cytologic samples is unusual and to date has only been reported in individuals who are crack (freebase cocaine) smokers. We report the cytologic finding of carbon-laden macrophages in pleural fluid unrelated to crack abuse. CASES: Two patients were identified with anthracotic pigment within their pleural fluid on cytologic review; an 88-year-old human immunodeficiency virus (HIV)-negative man with a transudative effusion and a 46-year-old HIV-positive man with a history of crack abuse who presented with an exudative effusion. Dense black pigment within macrophages was identified in both the ThinPrep slide and cell block material. This pigment failed to stain for iron and was present within the cytoplasm of KP-1 immunoreactive and TFF-1 negative macrophages. CONCLUSION: Carbon-laden macrophages can be found in exudative and transudative pleural effusions and may be seen without any relationship to crack abuse. Because this finding may be secondary to a subclinical pneumothorax, its identification and reporting may be of clinical significance.

Clinical practice: treatment of childhood empyema.

INTRODUCTION: The incidence of empyema in children is increasing. Adequate knowledge of treatment modalities is therefore essential for every pediatrician. At the university hospital of Leuven, the incidence per 100,000 admissions increased from 40 in 1993 to 120 in 2005. The treatment of choice, however, is still a matter of debate. This is mainly due to the scarcity of prospective randomized trials in children but is further complicated by the absence of uniform terminology. This review starts with clarifying definitions of empyema and complicated versus noncomplicated parapneumonic effusion. The place of different imaging techniques--ultrasound, chest X-ray, computerized tomography and magnetic resonance imaging--is illustrated. All treatment steps are evaluated starting with antibiotic choices, duration of i.v. and oral antibiotics, pleural fluid analysis, indications for chest drain placement, and fibrinolysis. As to the surgical interventions, there is at present insufficient evidence that early surgery is superior to noninvasive medical treatment. Therefore, video-assisted thoracoscopy cannot be advised as general first-line therapy. CONCLUSION: Since the pathogenicity of empyema is a dynamic process, therapeutic strategy must be decided based on empyema stage and clinical experience. Each referral center should agree on a diagnostic and therapeutic flowchart based on current evidence and local expertise. The flow chart outlined for our center is presented.

[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.

Cytokine levels in pleural effusions of patients under intensive care.

BACKGROUND: Pleural effusions develop for various reasons in patients admitted to intensive care units (ICUs). To understand why this occurs is important, yet cytokine levels in pleural effusions have rarely been measured from a cardiovascular viewpoint. OBJECTIVE: To understand the characteristics of pleural cytokines in patients admitted to the ICU. METHODS: The subjects were 43 patients with pleural effusion who were admitted to the ICU from June 2001 through March 2006. We divided the patients into transudate (n=23) and exudate (n=20) groups. We measured levels of interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha in pleural effusions and peripheral blood and evaluated their relationships with body temperature, C-reactive protein (CRP) level, and the peripheral white blood cell (WBC) count. RESULTS: Levels of pleural IL-6 were significantly higher and levels of TNF-alpha tended to be higher in pleural effusions from the exudate than in those from the transudate group (3,350+/-3,627 vs. 1,677+/-1,086 pg/m and 6.6+/-3.4 vs. 4.8+/-2.6 pg/mL, respectively). However, in both groups levels of IL-10 in pleural effusions were similar to those in serum and levels of IL-6 were significantly higher in pleural effusion than in serum. Serum IL-6 levels correlated with inflammatory markers (CRP and body temperature), whereas cytokines in pleural effusion did not correlate with any of these markers (body temperature, CRP, and WBC). CONCLUSION: Pleural levels of IL-6 were significantly higher in the exudate group than in the transudate group but did not correlate with serum levels of IL-6 or with systemic inflammatory markers. These findings suggest that pleural IL-6 levels correlate with local lung or pleural inflammation in patients admitted to the ICU.

Pleural mesothelial cells express both BLT2 and PPARalpha and mount an integrated response to pleural leukotriene B4.

Leukotriene B(4) (LTB(4)) plays a crucial role in the recruitment of neutrophils into the pleural space. We identified for the first time the mechanisms by which LTB(4) interacts with mesothelial cells and recruits neutrophils in the pleural compartment. Primary pleural mesothelial cells express both the proinflammatory receptor for LTB(4) BLT2, and the anti-inflammatory receptor for LTB(4), PPARalpha. Parapneumonic pleural effusions highly increase BLT2 expression and, via BLT2 activation, increase the adhesion between mesothelial cells and neutrophils and the expression of ICAM-1 on mesothelial cells. The block of PPARalpha further increases both cell adhesion and ICAM-1 expression. BLT2 activation promotes the activation, on mesothelial cells, of STAT-1 but not the activation of NF-kappaB transcription factor. The increase of ICAM-1 expression is achieved via increased tyrosine phosphorylation activity since herbimycin, a tyrosine kinase inhibitor, reduces and since Na orthovanadate, a tyrosine phosphatase inhibitor, further increases ICAM-1 expression. This study demonstrates that pleural mesothelial cells, expressing both proinflammatory and anti-inflammatory LTB(4) receptors, are able to mount an integrated response to LTB(4) with a prevalence of BLT2 activities in the presence of an inflammatory milieu within the pleura.

[Influence of the method used to obtain pleural fluid on the determination of the Acid-base balance]. / Importancia de la forma de obtención del líquido en la determinación del equilibrio ácido-base pleural.

OBJECTIVE: To analyze the methods used in our hospital for obtaining pleural fluid to determine the acid-base balance and to evaluate the clinical repercussions of each method. METHODS: Initially we studied the methods used by physicians in our hospital to collect pleural fluid for determination of the acid-base balance. In a second phase, we performed a prospective, descriptive, comparative study with the participation of 71 patients with pleural effusions in order to compare the acid-base balance according to the technique used to obtain the fluid. RESULTS: Pleural fluid was obtained using 3 methods: a) direct extraction using a heparinized syringe (group 1); b) extraction using a 20 mL syringe with subsequent aspiration from this syringe into a heparinized syringe (group 2); and c) filling a heparinized syringe from the 20 mL syringe (group 3). The only significant differences between group 1 and groups 2 and 3 were an increase in the pleural PO2 and oxygen saturation. The difference in the mean pH between groups 1 and 2 was 0.009 (95% confidence interval: -0.39 to 0.02; P=.5) and between groups 1 and 3 was 0.007 (95% confidence interval: -0.38 to 0.023; P=.6). The correlations between findings for PO2, pH, and PCO2 obtained in the different groups were statistically significant, with values superior to .95 in the last 2 variables. CONCLUSIONS: Physicians who perform thoracentesis in our hospital use different methods for obtaining fluid to determine the pleural acid-base balance. The 3 methods analyzed show no significant differences with regard to pH or PCO2. Pleural fluid may be obtained by a single puncture with a large-volume syringe, subsequently transferring the fluid to a heparinized syringe without this significantly affecting the pH or PCO2, thus reducing the number of manipulations and the risk of complications.

Immunocytochemical panel for distinguishing carcinoma cells from reactive mesothelial cells in pleural effusions.

OBJECTIVE: The aim of this study was to evaluate the individual and combined diagnostic utility of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CK19) and HBME-1 in pleural effusions of patients with lung cancer. STUDY DESIGN: CEA, CK19 and HBME-1 were detected by immunocytochemistry in pleural effusions from patients with lung cancer (86 cases) and without lung cancer (40 cases). RESULTS: CEA and CK19 expression were significantly higher in the carcinoma cell group and in three subgrouped as adenocarcinoma (AC), squamous cell carcinoma (SCC) and small cell lung cancer than in the mesothelial cell group, whereas HBME-1 expression was lower in the former group (P < 0.01). In the subgrouped tumours, CEA expression was higher in AC than in SCC (P < 0.05), whereas HBME-1 expression was higher in SCC than in AC (P < 0.01). Used alone, CK19 had the highest sensitivity (95.3%) and accuracy (93.7%), whereas CEA had the highest specificity (97.5%). When combinations of antibodies were evaluated together and membrane staining with HBME-1 taken as a negative outcome, CK19 and HBME-1 gave a high diagnostic performance: sensitivity of 100.0% and accuracy of 95.2% respectively. CONCLUSION: A panel of CEA, CK19 and HBME-1 monoclonal antibodies proved to be suitable for distinguishing carcinoma cells from reactive mesothelial cells in pleural effusions.

[Treatment indication with endothoracic drainage tube in parapneumonic effusions by partial pressure of carbon dioxide measurement in pleural fluid]. / Indicación de tratamiento con tubo de drenaje endotorácico en derrames pleurales paraneumónicos, según la presión parcial de anhídrido carbónico de líquido pleural.

BACKGROUND AND OBJECTIVE: Parapneumonic effusions with pH < 7.20 or glucose < 0.40 g/l or lactate dehydrogenase (LDH) > 1000 U/l have indication of treatment with endothoracic drainage tube (EDT). The aim of the present study was to determine the accuracy of partial pressure of carbon dioxide (pCO2) measurement in pleural fluid for the subsequent treatment indication with EDT in parapneumonic effusions, by analyzing the area under curve ROC (AUC) and determining the optimal cut off value. PATIENTS AND METHOD: 207 pleural fluids were studied. Glucose, LDH, pCO2, and pH were measured, and data concerning the etiology of pleural effusion and whether EDT treatment was needed were collected after patients were discharged from hospital. RESULTS: Forty-six out of 207 pleural fluids studied were parapneumonic effusions. Thirty-two were treated with EDT. AUC values were 0.888 (p < 0.0001), 0.890 (p < 0.0001), 0.816 (p < 0.0001), and 0.801 (p < 0.0001) for pCO2, pH, glucose, and LDH, respectively. No significant differences were found among them. Optimal cut off value for pCO2 was 48.6 mmHg, exhibiting 90.6% sensitivity and 78.6% specificity. All parapneumonic effusions showing pCO2 > 60.9 mmHg were treated with EDT. Remarkably, 3 out of 46 parapneumonic effusions (6.5%) that had been improperly treated following pH, glucose or LDH values, were correctly treated following pCO2. CONCLUSIONS: pCO2 determination in pleural fluid appears to be the best way to decide the indication of EDT in parapneumonic effusions.

[Laboratory diagnostics of pleural effusion]. / Uvod do laboratorní diagnostiky pleurálního výpotku.

Pleural effusion is a frequent reason for a pulmonologist's investigation. Many pulmonary and extrapulmonary causes of pleural effusion exist. Heart failure, pneumonia and malignancies are the most frequent among them. Laboratory examination of pleural liquid is a corner stone of diagnostics. We use various biochemical, microbiological, cytologic and other methods. The first step is a differentiation between transudate and exudate. If the laboratory examinations are unsuccessful, we can use invasive procedures - pleural biopsy and thoracoscopy. Despite all modern diagnostic methods the causes of about 15% pleural effusions remain unclear.

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.

A decision tree for differentiating tuberculous from malignant pleural effusions.

OBJECTIVE: To improve physicians' ability to discriminate tuberculous from malignant pleural effusions through a simple clinical algorithm that avoids pleural biopsy. DESIGN: We retrospectively compared the clinical and pleural fluid features of 238 adults with pleural effusion who satisfied diagnostic criteria for tuberculosis (n=64) or malignancy (n=174) at one academic center (derivation cohort). Then, we built a decision tree model to predict tuberculosis using the C4.5 algorithm. The model was validated with an independent sample set from another center that included 74 tuberculous and 293 malignant effusions (validation cohort). RESULTS: Among 12 potential predictor variables, the classification tree analysis selected four discriminant parameters (age>35 years, pleural fluid adenosine deaminase>38U/L, temperature>or=37.8 degrees C, and pleural fluid LDH>320U/L) from the derivation cohort. The generated flowchart had 92.2% sensitivity, 98.3% specificity, and an area under the ROC curve of 0.976 for diagnosing tuberculosis. The corresponding operating characteristics for the validation cohort were 85.1%, 96.9% and 0.958. CONCLUSIONS: Applying a decision tree analysis that contains simple clinical and laboratory data can help in the differential diagnosis of tuberculous and malignant pleural effusions.

Assessment of endoglin and calprotectin as potential biomarkers in ovarian carcinoma and borderline tumors of the ovary.

OBJECTIVE: The objective of the study was to analyze circulating endoglin concentration in ovarian carcinoma and evaluate a prognostic role for calprotectin and endoglin in effusions in advanced-stage disease. STUDY DESIGN: Preoperative plasma concentration of endoglin from women with benign ovarian tumors (n = 71), borderline ovarian tumors (BOT, n = 39), and ovarian carcinomas (n = 89) was analyzed with an enzyme-linked immunosorbent assay, as were endoglin and calprotectin concentrations in effusions from 164 women with advanced-stage ovarian carcinoma. RESULTS: Median endoglin plasma concentration was higher in the BOT group as compared with both control and invasive carcinoma groups (4.9 vs 4.5 and 4.3 ng/mL, P = .04 and P = .02), whereas the difference between the control and invasive group was not statistically significant (4.5 vs 4.3 ng/mL, P = .08). Endoglin and calprotectin effusion concentrations did not correlate with survival. CONCLUSION: Circulating endoglin is not elevated in advanced ovarian carcinoma. This is in contrast to the situation in breast and gastric cancer.

Clinically important factors influencing the diagnostic measurement of pleural fluid pH and glucose.

RATIONALE: Accurate pleural fluid pH and glucose measurement is a key component in the diagnosis and management of patients with pleural effusion. Standardized methods of pleural fluid collection have not been defined. OBJECTIVES: To assess the effect of common clinical factors that may distort measurement accuracy of pleural fluid pH and glucose. METHODS: Ninety-two exudative pleural aspirates were collected in commercially available blood gas syringes. MEASUREMENTS AND MAIN RESULTS: Samples were analyzed immediately using a blood gas analyzer. The effects of residual air, lidocaine, heparin, and delay in analysis (24 h) on pH and glucose measurement accuracy were assessed. Pleural fluid pH was significantly increased by residual air (mean +/- SD, 0.08 +/- 0.07; 95% confidence interval [CI], 0.06 to 0.09; P < 0.001) and significantly decreased by residual lidocaine (0.2 ml; mean change in pH, -0.15 +/- 0.09; 95% CI, -0.13 to -0.18; P < 0.001) and residual heparin (mean change in pH, -0.02 +/- 0.05; 95% CI, -0.01 to -0.04; P = 0.027). Pleural fluid pH was stable at room temperature for 1 hour and significantly increased at 4 (mean +/- SD, 0.03 +/- 0.07; 95% CI, 0.01 to 0.04; P = 0.003) and 24 hours (0.05 +/- 0.12; 95% CI, 0.03 to 0.08; P < 0.001). Pleural fluid glucose concentration was not clinically significantly altered by residual air, lidocaine (up to 0.4 ml), or 24-hour analysis delay. CONCLUSIONS: Accuracy of measured pleural pH is critically dependent on sample collection method. Residual air, lidocaine, and analysis delay significantly alter pH and may impact on clinical management. Pleural fluid glucose concentration is not significantly influenced by these factors. Protocols defining appropriate sampling and analysis methods are needed.

Categorisation of pleural fluids in routine clinical practice: analysis of pleural fluid protein and lactate dehydrogenase alone compared with modified Light's criteria.

Modified Light's criteria are widely used to categorise pleural fluids as either exudates or transudates. These criteria include fluid/serum ratios and therefore require a blood sample. It is not clear whether analysis of pleural fluid alone would alter pleural fluid categorisation in routine clinical practice. In this study, pleural fluids were categorised using cut-off values for pleural fluid protein (> or =30 g/l) and lactate dehydrogenase (>0.45 of upper limit of the serum reference interval) recommended as providing optimal discrimination between transudates and exudates. The resultant categorisations were compared with those produced by application of modified Light's criteria. 77 pleural fluid specimens were included for analysis. Using modified Light's criteria, 32 fluids were categorised as transudates and 45 as exudates. Applying the pleural fluid cut-off values as outlined above, 26 fluids were categorised as transudates and 51 as exudates. Agreement was observed in 71/77 or 92.2% (95% confidence interval 86.2 to 98.2). It was concluded that in most cases, analysis of pleural fluid protein and lactate dehydrogenase alone produces the same categorisation as modified Light's criteria. Omission of a blood sample rarely affects the categorisation of pleural fluids in routine clinical practice.

N-glycoprotein profiling of lung adenocarcinoma pleural effusions by shotgun proteomics.

BACKGROUND: Malignant pleural effusion of advanced lung adenocarcinoma may be a valid source for detection of biomarkers, such as N-glycosylated proteins (N-GP), because tumor cells grow during weeks in this liquid. The authors aimed for creation of N-GP effusion profiles from routine cytology specimens to detect relevant biomarkers. METHODS: Hundred microliters of malignant pleural effusions of 5 patients with lung adenocarcinoma and 5 nonmalignant controls were used for triplicate N-GP capture by solid-phase extraction. After trypsin digest and PNGase F release, a liquid chromatography separation connected online to a tandem mass spectrometer was performed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: In the total of 10 samples, 170 and 278 nonredundant proteins were detected with probabilities of >or=.9 and >or=.5, respectively. The specificity for the N-glycomotif was 88% at P >or= .9. Penetration into the moderate to low protein concentration range (microg-ng/mL) occurred, and several proteins associated with tumor progression or metastasis were identified, including CA-125, CD44, CD166, lysosome-associated membrane glycoprotein 2 (LAMP-2), multimerin 2, and periostin. MS identifications were correlated with the corresponding immunoreactivity in either effusion fluid or tumor tissue. CONCLUSIONS: In conclusion, reduction of sample complexity by N-GP capturing allows detection of proteins in the mug to ng/mL range. Pleural effusion is a useful source for biomarker research in lung cancer.