[Pre-analytical quality in fluid samples cytopathology: Results of a survey from the French Society of Clinical Cytology]. / Qualité pré-analytique en cytopathologie des liquides biologiques : résultats d'une enquête de la Société française de cytologie clinique.

INTRODUCTION: The pre-analytical step includes sample collection, preparation, transportation and storage in the pathology unit where the diagnosis is performed. The pathologist ensures that pre-analytical conditions are in line with expectations. The lack of standardization for handling cytological samples makes this pre-analytical step difficult to harmonize. Moreover, this step depends on the nature of the sample: fresh liquid or fixed material, air-dried smears, liquid-based cytology. The aim of the study was to review the different practices in French structures of pathology on the pre-analytical phase concerning cytological fluids such as broncho-alveolar lavage (BALF), serous fluids and urine. METHODS: A survey was conducted on the basis of the pre-analytical chapter of the ISO 15189 and sent to 191 French pathological structures (105 public and 86 private). RESULTS: Fifty-six laboratories replied to the survey. Ninety-five per cent have a computerized management system and 70% a manual on sample handling. The general instructions requested for the patients and sample identification were highly correctly filled with a short time routing and additional tests prescription. By contrast, information are variable concerning the clinical information requested and the type of tubes for collecting fluids and the volumes required as well as the actions taken in case of non-conformity. For the specific items concerning BALF, serous fluids and urine, this survey has shown a great heterogeneity according to sample collection, fixation and of clinical information. CONCLUSION: This survey demonstrates that the pre-analytical quality for BALF, serous fluids and urine is not optimal and that some corrections of the practices are recommended with a standardization of numerous steps in order to increase the reproducibility of additional tests such as immunocytochemistry, cytogenetic and molecular biology. Some recommendations have been written.

[Technical recommendations and best practice guidelines for May-Grünwald-Giemsa staining: literature review and insights from the quality assurance]. / Recommandations techniques et règles de bonne pratique pour la coloration de May-Grünwald-Giemsa : revue de la littérature et apport de l'assurance qualité.

May-Grünwald-Giemsa (MGG) stain is a Romanowsky-type, polychromatic stain as those of Giemsa, Leishman and Wright. Apart being the reference method of haematology, it has become a routine stain of diagnostic cytopathology for the study of air-dried preparations (lymph node imprints, centrifuged body fluids and fine needle aspirations). In the context of their actions of promoting the principles of quality assurance in cytopathology, the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP) and the French Society of Clinical Cytology (SFCC) conducted a proficiency test on MGG stain in 2013. Results from the test, together with the review of literature data allow pre-analytical and analytical steps of MGG stain to be updated. Recommendations include rapid air-drying of cell preparations/imprints, fixation using either methanol or May-Grünwald alone for 3-10minutes, two-step staining: 50% May-Grünwald in buffer pH 6.8 v/v for 3-5minutes, followed by 10% buffered Giemsa solution for 10-30minutes, and running water for 1-3minutes. Quality evaluation must be performed on red blood cells (RBCs) and leukocytes, not on tumour cells. Under correct pH conditions, RBCs must appear pink-orange (acidophilic) or buff-coloured, neither green nor blue. Leukocyte cytoplasm must be almost transparent, with clearly delineated granules. However, staining may vary somewhat and testing is recommended for automated methods (slide stainers) which remain the standard for reproducibility. Though MGG stain remains the reference stain, Diff-Quik(®) stain can be used for the rapid evaluation of cell samples.

Crizotinib Associated with Ground-Glass Opacity Predominant Pattern Interstitial Lung Disease: A Retrospective Observational Cohort Study with a Systematic Literature Review.

BACKGROUND: Crizotinib, an oral tyrosine kinase inhibitor that targets anaplastic lymphoma kinase, has proven to offer sustained progression-free survival in anaplastic lymphoma kinase-rearranged non-small-cell lung cancers. Occurrence of severe interstitial lung disease (ILD) was one of the crucial adverse events reported in randomized clinical trials and case reports. METHODS: In September 2011, we observed a crizotinib-associated ILD case. Following this index case, we reviewed the clinical and computed tomographic scan features of all patients treated with crizotinib in our department, between October 2010 and July 2013, comparing patients with and without ILD. A systematic literature review was performed. RESULTS: During this period, 29 patients were treated with crizotinib, five of whom developed ILD, in addition to the index case. Two types of adverse lung reactions may be observed in patients undergoing crizotinib therapy. The first is a severe, usually fatal, ILD that occurs during the first month of treatment (n = 1). The second is a less severe ILD, occurring later in time (n = 5). It occurs gradually with only few clinical symptoms, but predominant ground-glass opacities on computed tomography, along with an intensive lymphocytic alveolitis in bronchoalveolar lavage fluid. These cases had a longer response with a median progression-free survival duration at 19.9 months (17.9-23.5) compared with 6.2 months (1.2-13.6) for controls (p = 0.04). CONCLUSION: Forty-nine cases of crizotinib-associated ILD have been identified by the systematic review of the literature, including our six cases. Two types of adverse lung reactions may be observed with different presentation, prognosis, and treatment. Their potential mechanisms should be clarified. Nine patients with the less severe form of ILD were safely retreated.

The impact of intracytoplasmic mucin in lung adenocarcinoma with pneumonic radiological presentation.

OBJECTIVES: This study investigated the clinical and prognostic impact of intracytoplasmic mucin in lung adenocarcinoma with "pneumonic" radiological presentation, formerly known as bronchioloalveolar carcinoma (BAC). PATIENTS AND METHODS: Between 1986 and 2011, clinical and pathological data from 120 consecutive patients with lung adenocarcinoma with "pneumonic" radiological presentation were reviewed. Intracytoplasmic mucin was assessed using a diastase-resistant periodic acid-Schiff staining. The presence of EGFR or K-Ras mutations and ALK rearrangement were determined in surgical samples. RESULTS: The two predominant histological patterns were invasive mucinous adenocarcinoma (40%) and lepidic predominant adenocarcinoma (32%). Intracytoplasmic mucin was detected in 71 patients (59.2%) who were more likely to be non-smokers (p=0.04) and have bronchorrhea (p=0.006), crepitant rales (p=0.02), or neutrophil alveolitis (p=0.0004). In mucin-producing tumors, EGFR mutation was not detected, K-Ras mutations and ALK rearrangement were present in 32% and 3% of cases, respectively. In non-mucin-producing tumors, EGFR and K-Ras mutations were detected in 17% and 10% of cases, respectively, no ALK rearrangement was detected. In univariate analysis, performance status>0, crepitant rales, bronchorrhea, neutrophil alveolitis, bilateral extension, intracytoplasmic mucin and no surgery were associated with worse survival. In multivariate analyses, intracytoplasmic mucin, neutrophil alveolitis, and no surgery were independent factors for worse survival. CONCLUSION: Intracytoplasmic mucin is associated with specific clinical characteristics and is an independent factor for worse survival in lung adenocarcinoma formerly known as BAC.

Cytological specimens obtained by endobronchial ultrasound-guided transbronchial needle aspiration: sample handling and role of rapid on-site evaluation.

Recently developed, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive modality for mediastinal lymph node staging in lung cancer patients as well as for the diagnosis of mediastinal and hilar lymphadenopathy. It has been shown in systematic reviews and meta-analysis that a high diagnostic yield can be achieved with EBUS-TBNA for staging lung cancer. Though still not a standard of practice, this novel technology has attracted physicians and surgeons as an alternative modality to surgical biopsy for the assessment of the mediastinum. Standard cytology, thin layer preparations in liquid medium or cell blocks of cells obtained by EBUS-TBNA can be applicable not only for pathological diagnosis but also for further investigations such as immunohistochemistry and fluorescence in situ hybridization. In addition, samples obtained by EBUS-TBNA can also be used for molecular analysis. The key to a successful EBUS-TBNA is to understand the anatomy of the mediastinum as well as the basic steps of the procedure. Moreover, handling of the sample obtained by EBUS-TBNA is crucial for diagnosis since no amount of skill or interest of the interpreter can make up for a badly prepared sample. The goals of rapid on-site evaluation during EBUS-TBNA include determination of whether sampling of the target has been achieved and more importantly triage of samples to secondary investigations. This manuscript explains the detailed techniques of EBUS-TBNA to master this innovative procedure.

A comparative transmission electron microscopy study of titanium dioxide and carbon black nanoparticles uptake in human lung epithelial and fibroblast cell lines.

Several studies suggest that the biological responses induced by manufactured nanoparticles (MNPs) may be linked to their accumulation within cells. However, MNP internalisation has not yet been sufficiently characterised. Therefore, the aim of this study was to compare the intracellular uptake of three different MNPs: two made of carbon black (CB) and one made of titanium dioxide (TiO(2)), in 16HBE bronchial epithelial cells and MRC5 fibroblasts. Transmission electron microscopy was used to evaluate the intracellular accumulation. Different parameters were analysed following a time and dose-relationship: localisation of MNPs in cells, percentage of cells having accumulated MNPs, number of aggregated MNPs in cells, and the size of MNP aggregates in cells. The results showed that MNPs were widely and rapidly accumulated in 16HBE cells and MRC5 fibroblasts. Moreover, MNPs accumulated chiefly as aggregates in cytosolic vesicles and were absent from the mitochondria or nuclei. CB and TiO(2) MNPs had similar accumulation patterns. However, TiO(2) aggregates had a higher size than CB aggregates. Intracellular MNP accumulation was dissociated from cytotoxicity. These results suggest that cellular uptake of MNPs is a common phenomenon occurring in various cell types.

Clonality and phenotyping analysis of alveolar lymphocytes is suggestive of pulmonary MALT lymphoma.

BACKGROUND: Mucosa-associated lymphoid tissue (MALT) lymphoma, a low-grade B-cell extranodal lymphoma, is the most frequent subset of primary pulmonary lymphoma (PPL). It often associates with connective tissue disease (CTD). We aimed to evaluate the impact of concomitant CTD on diagnostic value of flow cytometry and genetic clonality analyses for the diagnostic of MALT lymphoma. METHODS: All chest disease and pathology departments of teaching hospitals in Paris were contacted to identify patients with a histological diagnosis of PPL of the MALT subtype with or without associated CTD. We identified 44 patients in the lymphoma group; 11 had a CTD and were matched to 11 patients with CTD but without lymphoma. RESULTS: Results of BAL analyses of MALT-PPL showed normal cellularity (370 cells/mm(3) [range 21-2300]) but increased proportion of lymphocytes (31.5% [80-2]) of the B-cell subtype (20% [1-88]). A B-cell clone was detected in 82% of cases, and specificity of clonality was 90%. Interestingly, BAL analysis results different by presence or not of a CTD. The frequency of B lymphocyte alveolitis was significantly greater in MALT patients without than with CTD (34% vs 6.5%, p = 0.007). However, BAL results for patients with CTD did not differ between those with and without lymphoma. CONCLUSION: BAL results may be highly suggestive of pulmonary MALT lymphoma. The proportion of B-cells may vary depending on the presence of an associated CTD, but clonality analyses remained informative for the diagnostic of MALT lymphoma.

Syntenic relationships between genomic profiles of fiber-induced murine and human malignant mesothelioma.

Malignant mesothelioma (MM) is an aggressive tumor with a poor prognosis mainly linked to past asbestos exposure. Murine models of MM based on fiber exposure have been developed to elucidate the mechanism of mesothelioma formation. Genomic alterations in murine MM have now been partially characterized. To gain insight into the pathophysiology of mesothelioma, 16 murine and 35 human mesotheliomas were characterized by array-comparative genomic hybridization and were screened for common genomic alterations. Alteration of the 9p21 human region, often by biallelic deletion, was the most frequent alteration in both species, in agreement with the CDKN2A/CDKN2B locus deletion in human disease and murine models. Other shared aberrations were losses of 1p36.3-p35 and 13q14-q33 and gains of 5p15.3-p13 regions. However, some differences were noted, such as absence of recurrent alterations in mouse regions corresponding to human chromosome 22. Comparison between altered recurrent regions in asbestos-exposed and non-asbestos-exposed patients showed a significant difference in the 14q11.2-q21 region, which was also lost in fiber-induced murine mesothelioma. A correlation was also demonstrated between genomic instability and tumorigenicity of human mesothelioma xenografts in nude mice. Overall, these data show similarities between murine and human disease, and contribute to the understanding of the influence of fibers in the pathogenesis of mesothelioma and validation of the murine model for preclinical testing.

Excess of neuromuscular spindles in a fetus with Costello syndrome: a clinicopathological report.

The neuromuscular spindle (NMS) is a proprioceptive myofibrillar component of skeletal muscles that is necessary to maintain normal muscle tone and coordination. Recently, an excess of NMS has been reported as a congenital neuromuscular syndrome with a Noonan phenotype, now linked to Costello syndrome (CS). The vast majority of patients with CS have a de novo heterozygous mutation in the HRAS gene involved in the Ras/mitogen-activated protein kinase (MAPK) pathway. CS has many features in common with Noonan and cardiofaciocutaneous syndromes, also linked to activating mutations (but in other genes) of the Ras/MAPK pathway. This makes the orientation of molecular screening difficult. The observation of excess NMS in a 26-weeks'-gestation stillborn prompted us to screen the HRAS gene for mutation. The identification of a HRAS mutation made it possible to establish a diagnosis of CS. We conclude that the excess of NMS is the most reliable sign for the diagnosis of CS. Our findings also show the instrumental role of histological study of the skeletal muscles in the context of polyhydramnios and fetal hydrops.

Release of metal particles from needles used for transbronchial needle aspiration.

BACKGROUND: Although mediastinoscopy is still the gold standard for diagnosis of mediastinal lymphadenopathy, minimally invasive procedures have been developed: transbronchial needle aspiration (TBNA) using a flexible bronchoscope (conventional TBNA) or linear echoendoscope (endobronchial ultrasound [EBUS]) allowing real-time guided lymph node aspiration. The observation of contamination of samples by foreign particles led us to determine the frequency and the nature of this material and to identify its origin. METHODS: From June 2007 to November 2008, 141 consecutive patients underwent conventional TBNA (n = 84) or EBUS-guided TBNA (EBUS-TBNA) (n = 57). All cytologic samples were reviewed in blinded fashion, and contamination was assessed semiquantitatively. Mineral analysis using a transmission electron microscope equipped with an energy dispersive x-ray spectrometer was performed on the solution obtained after rinsing unused needles and on four samples of calf thymuses punctured with EBUS needles. RESULTS: Foreign material, different from anthracosis, was identified in samples obtained with five different batches of needles, only from EBUS-TBNA (P < .0001). The contamination score was correlated to the number of passes (P = .035). Mineral analyses of the rinsing solutions from conventional TBNA needles were negative, whereas metal alloys of iron, titanium, nickel, and chromium were released with EBUS needles. The same contamination was identified in three of the four punctured calf thymuses. CONCLUSIONS: Dedicated EBUS-TBNA needles are able to release metal particles, probably by friction between the stylet and the needle, with a potential risk to inject particles into nodes. The long-term consequences are unknown, but the need for safety measures should be evaluated.

Oxidative stress and proinflammatory effects of carbon black and titanium dioxide nanoparticles: role of particle surface area and internalized amount.

The ubiquitous presence of nanoparticles (NPs) together with increasing evidence linking them to negative health effects points towards the need to develop the understanding of mechanisms by which they exert toxic effects. This study was designed to investigate the role of surface area and oxidative stress in the cellular effects of two chemically distinct NPs, carbon black (CB) and titanium dioxide (TiO(2)), on the bronchial epithelial cell line (16HBE14o-). CB and TiO(2) NPs were taken up by 16HBE cells in a dose-dependent manner and were localized within the endosomes or free in the cytoplasm. Oxidative stress produced inside the cell by NPs was well correlated to the BET surface area and endocytosis of NPs. Contrary to intracellular conditions only CB NPs produced reactive oxygen species (ROS) under abiotic conditions. Exposure of cells to NPs resulted in an increased granulocyte macrophage colony stimulating factor (GM-CSF) mRNA expression and secretion. Inflammatory effects of NPs were dependent on the surface area and were mediated through oxidative stress as they were inhibited by catalase. It can be concluded that NP induced oxidative stress and pro-inflammatory responses are well correlated not only with the BET (Brunauer, Emmett and Teller) surface of the individual NPs but also with the internalized amount of NPs. Differences of even few nanometers in primary particle size lead to significant changes in inflammatory and oxidative stress responses.

In vitro effects of nanoparticles on renal cells.

BACKGROUND: The ability of nanoparticles to cross the lung-blood barrier suggests that they may translocate to blood and to targets distant from their portal of entry. Nevertheless, nanotoxicity in organs has received little attention. The purpose of this study was to evaluate nanotoxicity in renal cells using in vitro models. Various carbon black (CB) (FW2-13 nm, Printex60-21 nm and LB101-95 nm) and titanium dioxide (TiO2-15 and TiO2-50 nm) nanoparticles were characterized on size by electron microscopy. We evaluated theirs effects on glomerular mesangial (IP15) and epithelial proximal tubular (LLC-PK1) renal cells, using light microscopy, WST-1 assay, immunofluorescence labeling and DCFH-DA for reactive oxygen species (ROS) assay. RESULTS: Nanoparticles induced a variety of cell responses. On both IP15 and LLC-PK1 cells, the smallest FW2 NP was found to be the most cytotoxic with classic dose-behavior. For the other NPs tested, different cytotoxic profiles were found, with LLC-PK1 cells being more sensitive than IP15 cells. Exposure to FW2 NPs, evidenced in our experiments as the most cytotoxic particle type, significantly enhanced production of ROS in both IP15 and LLC-PK1 cells. Immunofluorescence microscopy using latex beads indicated that depending on their size, the cells internalized particles, which accumulated in the cell cytoplasm. Additionally using transmission electronic microscope micrographs show nanoparticles inside the cells and trapped in vesicles. CONCLUSION: The present data constitute the first step towards determining in vitro dose effect of manufactured CB and TiO2 NPs in renal cells. Cytotoxicological assays using epithelial tubular and glomerular mesangial cell lines rapidly provide information and demonstrated that NP materials exhibit varying degrees of cytotoxicity. It seems clear that in vitro cellular systems will need to be further developed, standardized and validated (relative to in vivo effects) in order to provide useful screening data about the relative toxicity of nanoparticles.

Neutrophils promote aerogenous spread of lung adenocarcinoma with bronchioloalveolar carcinoma features.

PURPOSE: Adenocarcinoma with bronchioloalveolar carcinoma (BAC) features is a subtype of non-small cell lung cancers characterized by an intense inflammatory reaction composed of macrophages and neutrophils and by a distinct natural history with intrapulmonary spread leading to death due to respiratory failure. We hypothesized that neutrophils could promote aerogenous spread of lung adenocarcinoma with BAC features. EXPERIMENTAL DESIGN: We examined the effect of neutrophils on A549 cell line detachment in vitro and we quantified desquamation of tumor cells on tumor tissue (n = 25) and on matched bronchioloalveolar lavage (n = 17) in vivo in a series of patients with adenocarcinoma with BAC features. RESULTS: Neutrophils induced A549 detachment mediated by signals through cell-to-cell contact. Detached A549 cells were still viable and able to proliferate in vitro. Neutralization studies identified several membrane-bound molecules involved in detachment (i.e., intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, tumor necrosis factor alpha/tumor necrosis factor alpha receptor inhibitor, interleukin-1alpha /interleukin-1alpha receptor, and neutrophil elastase). In tumor tissue, shedding was detected in all samples, with a median shedding score of 42% (range, 4-95%). Micropapillary clusters were detected in 23 of the 25 tumor tissue samples, with a median micropapillary score of 1.40 (range, 0-2.1), and tumor cells were detected in 7 of 17 lavages. The micropapillary score was associated with a high neutrophil count in bronchioloalveolar lavage (P = 0.051). The shedding cell percentage was a significant factor in shorter survival (P = 0.034, univariate Cox analysis). CONCLUSIONS: Tumor shedding is induced by neutrophils. It is a significant factor of shorter survival and may be an important event in adenocarcinoma progression.

Immunolocalization and cell expression of lung resistance-related protein (LRP) in normal and tumoral human respiratory cells.

Lung resistance-related protein (LRP) is an integral part of the multidrug resistance (MDR) phenotype involved in cell resistance toward xenobiotics or chemotherapy. The aim of this study was to compare the intracellular localization and cell expression of LRP in normal bronchial cells and their tumoral counterparts from non-small cell lung cancer (NSCLC). LRP expression was also investigated concurrently with DNA ploidy and chromosome 16 (lrp gene locus) aberrations. Confocal microscopy showed that LRP localization was exclusively intracytoplasmic regardless of the cell type and was never observed in the nuclear pore complex. Flow cytometry demonstrated a similar level of LRP expression in normal bronchial cells and in cancer cells from NSCLC samples. FISH analysis, performed to evaluate the number of chromosome 16 and lrp loci, demonstrated a significant gain of chromosome 16 in DNA aneuploid tumors. Furthermore, we did not find any link between LRP expression and DNA ploidy status or chromosome 16 number. These results suggest that LRP expression observed in NSCLC, maintained through the carcinogenesis process of respiratory cells, is not altered by the increased number of copies of chromosome 16 and probably controlled by mechanisms different from those of MRP1 expression, whereas both proteins are associated with the MDR phenotype.