Detection of Candida antigen in bronchoalveolar lavage fluid

While bronchoalveolar lavage is frequently performed to evaluate immunocompromised hosts for infection, the significance of rare yeasts found on the cytologic examination of lavage fluid is unclear. This study used the latex agglutination method to test lavage fluids for Candida antigen to assess its usefulness in distinguishing Candida pneumonia from Candida colonization of the respiratory tract or oral contamination of the lavage specimen. Ninety-seven specimens from 87 patients were categorized on the basis of historical, microbiologic, cytologic and serologic data. Bronchoalveolar lavage fluids were positive for Candida antigen in 0 of 20 specimens from normal controls, 0 of 14 specimens from patient controls, 5 (36%) of 14 specimens from patients with Pneumocystis carinii pneumonia, 0 of 5 specimens from patients with gastrointestinal candidiasis, 0 of 9 specimens contaminated by oral-derived yeasts, 2 (10%) of 19 specimens from patients with probable Candida colonization and 15 (94%) of 16 specimens from patients with clinical and laboratory evidence of Candida pneumonia. We conclude that this test assists in the differentiation of Candida pneumonia from other situations in which yeasts are recovered by bronchoalveolar lavage.     

Hemosiderin-laden macrophages in bronchoalveolar lavage fluid.

Diffuse pulmonary hemorrhage syndromes occasionally present diagnostic problems if the clinical picture is not very typical. The presence of hemosiderin-laden alveolar macrophages in bronchoalveolar lavage (BAL) fluid is a useful diagnostic criterion for this entity. However, in many diffuse interstitial pulmonary diseases (DIPD) there is a lesion at the alveolocapillary barrier, and an exit of red blood cells could occur from the blood vessels, leading to the appearance of siderophages. The aim of this work was dual: to evaluate the presence and number of siderophages in different types of interstitial pulmonary disease and to compare the diagnostic yield of two ways of quantifying hemosiderin-laden macrophages. Three groups of patients--controls (n = 5), DIPD (n = 32) and diffuse pulmonary hemorrhage (n = 3)--were subjected to BAL, and a differential count was made on cytocentrifuged Diff-Quik- and Perl-stained preparations. On the latter, two different measurements were made: the number of macrophages laden with hemosiderin and a quantitative "score." The results of a conventional count (percent of Perl-positive macrophages) showed significant differences between the three groups considered overall. Applying a cutoff value of 20%, the sensitivity of this method was 100% and the specificity, 91.6%. The results of the hemosiderin score showed significant differences between the three groups. Applying a value of 50 as a cutoff, the sensitivity and specificity of the method were 100%. In control patients and carriers of DIPD, the percentage of alveolar macrophages was higher than in healthy subjects. Quantification of the hemosiderin content of alveolar macrophages improved the specificity of the diagnosis of diffuse pulmonary hemorrhage by BAL.     

Familial pulmonary alveolar microlithiasis diagnosed by bronchoalveolar lavage. A case report

BACKGROUND: Familial alveolar microlithiasis is a rare lung disease. In this study we describe the cytologic features of this disease in bronchoalveolar lavage. CASE: A 10-year-old girl and her uncle, a 50-year-old man, had dyspnea and diffuse interstitial pattern on chest radiograph with no defined cause at a period of 10 years apart. Open lung biopsy in the girl and transbronchial lung biopsy plus bronchoalveolar lavage (BAL) in the man were per-formed to determine the diagnosis. In cyopen lung biopsy the diagnosis was alveolar microlithiasis. BAL revealed rehtypical microliths (calcospherites), and th transbronchial lung biopsy performed in the same patient failed to disclose superficially reset any significant pathology. In cytologic a smears, extracellular and intracellular concentrically layered purple-brown, round-to-oval microliths were clearly seen. Cyanophilic periodic acid-Schiff positive intracytoplasmic amorphous material was also frequently seen in alveolar macrophages. CONCLUSION: Familial alveolar microlithiasis is a rare interstitial lung disease that can be easily diagnosed by BAL. This procedure is a very useful tool in diagnosing and classifying some interstitial lung diseases.     

Proteomics of bronchoalveolar lavage fluid

Lung diseases are essentially multi-factorial diseases that require a global analysis, and thus, cannot be understood through the sole analysis of individual or small numbers of genes. Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the obligated complementary technology for genetic profiling. It has been shown to be a powerful tool for the study of human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. During last years, proteomic approaches applied to lung diseases are centred on the analysis of proteins in samples, such as cell cultures, biopsies and physiological fluids like serum and, especially, bronchoalveolar lavage fluid (BALF). BALF is presently the most common way of sampling the components of the epithelial lining fluid (ELF) and the most faithful reflect of the protein composition of the pulmonary airways. This review focuses on the state of the investigations of BALF proteome and its powerful contribution both to a better knowledge of the lung structure at the molecular level and to the study of lung disorders at the clinical level.     

Galactomannan detection in bronchoalveolar lavage fluid

Prompt diagnosis of invasive pulmonary aspergillosis (IPA) remains a challenge. Galactomannan (GM) assay in serum has been incorporated into diagnostic criteria for IPA, but its performance varies depending on the population in which the test is used. GM assay on bronchoalveolar lavage (BAL) fluid aims to improve upon the test by applying it directly on samples from the target organ. The studies that have examined the utility of BAL-GM are a heterogeneous group, but the results are intriguing, especially in patients who are at risk for IPA from causes other than hematologic malignancy and neutropenia. BAL-GM had sensitivities ranging from 60% to 100% in this group, often far exceeding the performance of serum GM assay. The test shows promise as a useful adjunctive diagnostic modality in the diagnosis of IPA.     

Penicillium marneffei in bronchoalveolar lavage fluid

Penicillium marneffei is a rare human pathogen that may infect either healthy or immunocompromised hosts. In methenamine silver-stained preparations of bronchoalveolar lavage fluid from a patient with dermatomyositis on steroid treatment, round-to-oval intracellular and extracellular microorganisms were found. The finding of occasional septate and elongated forms established the microorganism as probably P marneffei, which was confirmed on culture. Distinguishing this rare fungus from Pneumocystis carinii is important because these two diseases require different forms of treatment.     

Database of bronchoalveolar lavage fluid proteins

Bronchoalveolar lavage during fiberoptic bronchoscopy is extensively used for investigating cellular and biochemical alterations of the epithelial lining fluid in various lung disorders. Two-dimensional electrophoresis (2-DE) offers the possibility to simultaneously display and analyze proteins contained in bronchoalveolar lavage fluid (BALF). We present the current status of 2-DE of BALF samples with an updated listing of the proteins already identified and of their level and/or posttranslational alterations in lung disorders. Alternatives to 2-DE of BALF samples and future prospects of proteomics to unravel lung functions and pathologies are discussed.     

Detection of lipid peroxidation in lung and in bronchoalveolar lavage cells and fluid

Inhalation of toxic materials such as asbestos, silica, 100% oxygen, ozone, or nitrogen dioxide may lead to an increased production of reactive oxygen metabolites which may initiate lipid peroxidation. Measurement of lipid peroxidation in cells and fluid obtained by bronchoalveolar lavage (BAL), as well as in lung tissue, may aid in monitoring the development and extent of pulmonary damage after inhalation of a toxic substance. In this study, we employed a sensitive assay for detection of malondialdehyde (MDA), a breakdown product of lipid peroxidation. By separation of the adduct with thiobarbituric acid, using a reverse phase high pressure liquid chromatographic technique, we accurately and sensitively measured the content of MDA in BAL cells, lavage fluid, and lavaged lung tissue homogenates of rats. The amounts of sample required for detection of MDA were small enough possibly to be applied to use with human specimens; in addition, recovery of added MDA was acceptable with all types of samples. Inclusion of a metal chelator in the preparation of samples appeared necessary to prevent metal-catalyzed propagation of lipid peroxidation during the assay. Overall, the method described here using samples from rats may be applicable to detecting lipid peroxidation in BAL samples from humans.     

Diffuse alveolar damage. Morphologic features in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the overall cytologic characteristics of diffuse alveolar damage (DAD) in bronchoalveolar lavage (BAL) specimens in search of features that could be useful in cytologic diagnosis. STUDY DESIGN: We evaluated BAL samples from patients with DAD obtained simultaneously with transbronchial biopsies (n = 8) or open lung biopsies (n = 2) or within 24 hours of autopsy (n = 2). The material was processed routinely for cytologic and histologic evaluation. RESULTS: The smears were moderately to highly cellular. All cases had large numbers of alveolar macrophages and/or desquamated alveolar cells. The epithelial component displayed various degrees of nuclear atypia. Some epithelial clusters were three-dimensional, with peripheral cells showing clear cytoplasm, protruding outwards and resembling hobnails. Other aggregates appeared two-dimensional, as sheets of cells with flattened and dense cytoplasm (squamotized). Both types of cell clusters were often associated with dense, basophilic or amphophilic, amorphous extracellular material. Counterparts of all the cytologic features were observed in the histologic material, including atypia of the alveolar lining with hobnailing, squamotization, amorphous extracellular material and hyaline membranes. CONCLUSION: The cytologic features of BAL represent a constellation of alveolar cell injury. Based on these features, DAD can be correctly diagnosed or suggested in BAL samples in the appropriate clinical setting.     

Reactive type II pneumocytes in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the prevalence of reactive type II pneumocytes (RPII) in bronchoalveolar lavage (BAL) fluid samples obtained from patients with various pulmonary disorders. STUDY DESIGN: Consecutive BAL fluid samples were screened for the presence of RPII on May-Grünwald-Giemsa-stained cytocentrifuge preparations. BAL fluid samples with and without RPII were compared with regard to prevalence, associated clinical diagnoses and cytologic findings. RESULTS: RPII were generally large cells with a high nuclear:cytoplasmic ratio and deeply blue-stained, vacuolated cytoplasm. Most RPII occurred in cohesive cell groups, and the vacuoles tended to be confluent. Cytologic findings associated with RPII were foamy alveolar macrophages, activated lymphocytes and plasma cells. RPII were present in 94 (21.7%) of 433 included BAL fluid samples. The highest prevalences were noted in patients with systemic inflammatory response syndrome and alveolar hemorrhage. In addition, RPII tended to occur more frequently in ventilator-associated pneumonia, Pneumocystis carinii pneumonia, extrinsic allergic alveolitis and drug-induced pulmonary disorders. In contrast, RPII were not observed in BAL fluid samples obtained from patients with sarcoidosis. CONCLUSION: RPII were prevalent in about 20% of BAL fluid specimens. They were associated mainly with conditions of acute lung injury and not observed in sarcoidosis.     

Use of poly-l-lysine-coated slides in clinical bronchoalveolar lavage fluid samples

OBJECTIVE: Polylysine coating of microscope slides provides superior cell adhesion. We compared poly-l-lysine-coated (PLC) slides to conventional slides in cytocentrifuged bronchoalveolar lavage (BAL) fluid samples. STUDY DESIGN: Twenty BAL fluid samples with representative numbers of alveolar macrophages, lymphocytes and polymorphonuclear neutrophils were cytocentrifuged on uncoated slides and on PLC slides (2 slides each). Cell density, differential cell counts and cytomorphology were assessed on May-Grünwald-Giemsa-stained preparations. Reliability of cell differentiation was expressed as a phi value, which measures combined reproducibility and agreement. Statistical significance of differences between slides was calculated with ANOVA. Clinical relevance was assessed using a validated computer program predicting the most probable diagnosis. RESULTS: Although not statistically significant, cell recovery was lower on PLC slides as compared to uncoated slides. PLC slides held significantly fewer lymphocytes as compared to uncoated slides (mean value +/- SD: 25.89% +/- 28.26 versus 28.34% +/- 29.96, respectively). Counts of alveolar macrophages, lymphocytes and polymorphonuclear neutrophils displayed excellent phi values for both uncoated and PLC slides. No discrepancies in the computer-generated diagnoses were found. CONCLUSION: For BAL fluid cytology on cytocentrifuged preparations, PLC slides are not superior to conventional slides.     

Detection of disease specific sialoglycoconjugate specific antibodies in bronchoalveolar lavage fluid of non-small cell lung cancer patients

In this study, we report the presence of significantly higher level of GM3 specific IgG antibodies (IgG_TL) in the bronchoalveolar lavage fluid obtained from tumor bearing lung of non-small cell lung cancer (NSCLC) patients as compared to other non-neoplastic controls. The antibodies were isolated using DEAE-cellulose anion exchange chromatography and molecular weight of the subunits of IgG_TL was confirmed in SDS-PAGE. IgG_TL revealed high specificity to GM3 and the IgG distribution was confined to IgG1. Furthermore, IgG_TL showed strong reactivity with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. A 66 kDa membrane glycoprotein of NSCLC cell lines was found to interact specifically with IgG_TL, the intensity of which was drastically reduced in presence of GM3. Further, binding of Maackia amurensis agglutinin [specific for NeuAcα(2→3)Gal unit , the same disaccharide unit also known to be present in GM3] to the 66 kDa band confirmed it to be a sialoglycoprotein in nature. IgG_TL could not show any reactivity to alkaline borohydrate treated or periodate oxidised membrane fractions, suggesting the probable involvement of the carbohydrate moiety of the 66 kDa glycoprotein in the interaction with IgG_TL. Thus, the 66 kDa sialoglycoprotein seems to be the NSCLC specific sialoglycoconjugate. Taken together, IgG_TL antibodies may have the potential to serve as a unique probe for detail investigation of NSCLC specific cell surface sialoglycoconjugate. Further, due to high specificity of IgG_TL to GM3, it may be possible to develop a simple alternative diagnostic approach (GM3-ELISA) for NSCLC.     

Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs

We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n = 12)- and endotoxin (n = 18)-treated pigs. Endotoxin, infused at 5 micrograms/kg for 1 hr followed by 2 micrograms/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia. Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 +/- 209 pg/ml), i-12-HETE (1589 +/- 517 pg/ml), and i-15-HETE (448 +/- 78 pg/ml) were significantly (P less than 0.05) increased in BALF recovered from endotoxemic pigs at postmortem. Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively. We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.     

Quantitation of surfactant protein B by HPLC in bronchoalveolar lavage fluid

A sensitive reversed phase HPLC method with evaporative light scattering detection (ELSD) was developed for the determination of the hydrophobic surfactant protein B (SP-B) in human bronchoalveolar lavage fluid. Samples were extracted two times with CHCl(3):MeOH:HCl (2:3:0.005N) solution in a ratio of 1:2 by volume. The extract of the lower phase was separated on a C4 butyl silica gel column with an isocratic elution using a mobile phase, consisting of 97% methanol, 2.75% chloroform and 0.25% 0.1 M trifluoroacetic acid (by volume), at a flow rate of 1 ml/min. SP-B was detected by ELSD and quantified by comparison to an external standard. The duration of a run was 7 min, the quantification limit 30 ng and the limit of detection was at about 15 ng of SP-B. This method is suitable for the rapid routine quantification of SP-B in human bronchoalveolar lavage fluid samples.     

Use of a fluid dispensing apparatus for bronchoalveolar lavage in mice

The volume of the mouse lung is small, so bronchoalveolar lavage (BAL) in mice is generally performed with 1 ml syringes to infuse smaller volumes of fluid. Multiple infusions are required to obtain enough recovered fluid for multiple analyses. This paper introduces the use of one type of a simple fluid dispensing apparatus as an infusion device. It proved to be a faster and a less tedious method than the syringe infusion method. The results of studies in normal mice using both infusion techniques showed no differences between the two with respect to the recovery of cells and protein and to differential leukocyte counts. Thus, the results obtained with this device can be compared with those previously obtained with syringes.     

Elastolytic activity of bronchoalveolar lavage fluid in acute lung inflammatory injury

Elastolytic activity in bronchoalveolar lavage fluid in the lung with acute inflammatory injury and properties of different proteinase inhibitors for its correction was established. It was determined, that 4/5 of elastolytic activities are submitted to neutrophile serine proteinase (EC 3.4.21.37) and 1/5 of elastolytic activities - metalloenzymes macrophages origin (EC 3.4.24.65). Inhibition of elastase-like activity with the use of three proteinase inhibitors: contrycal, ingiprol and thermo- and acid-stable proteinase inhibitor from rabbit blood showed more intensive ability of thermo- and acid-stable proteinase inhibitor to inhibit pancreatic elastase and pull of neutrophil and macrophage elastase. Preventive use and treatment of proteinase inhibitors effectively suppressed activation of proteinases in the acute lung inflammatory injury.     

Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs

We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n=12)- and endotoxin (n=18)- treated pigs. Endotoxin, infused at 5 μg/kg for 1 hr followed by 2 μg/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia. Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 ± 209 pg/ml), i-12-HETE (1589 ± 517 pg/ml), and i-15-HETE (448 ± 78 pg/ml) were significantly ) increased in BALF recovered from endotoxemic pigs at postmortem. Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively. We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.