Association of Stress Coping Strategies with Immunological Parameters in Melanoma Patients.

In this exploratory case control study the association between stress coping strategies and lymphocyte subpopulations was calculated in 18 non-metastatic melanoma patients and 18 controls with benign skin diseases. Coping strategies were assessed using the German version of the stress-coping questionnaire (SVF 120). While in the control group patients showed significant negative correlations of lymphocyte subpopulations (CD3+, CD4+, CD8+, CD19+, CD45+ cells) with coping strategies that refer to defence, in melanoma patients significant positive correlations between lymphocyte subpopulations (CD3+, CD4+, CD19+, CD45+ cells) were found with regard to coping strategies that are characterized by diversion from stress and focusing on stress-compensating situations. The present data, in melanoma patients and controls, show contrary correlations between stress coping strategies and lymphocyte subpopulations. The interconnection between stress coping and immunologic alterations in malignant melanoma is a field deserving further multiprofessional investigation in order to provide new therapeutical approaches in the treatment and understanding of melanoma patients.

Psychological Stress and Immunological Modulations in Early-stage Melanoma Patients.

Mental stress may have a negative impact on the immune state of cancer patients, in whom immunologic surveillance is essential for survival. This study investigated the immunological response of 19 patients with early-stage melanoma and a matched control group undergoing the Determination Stress Test before surgery. Cytokine and chemokine levels and lymphocyte subpopulations were measured at baseline and post-stress test time-points. Following the stress test lower levels of interleukin (IL)-6 were observed in the melanoma group compared with healthy volunteers (p = 0.044). IL-10 increased significantly in the control group 30 min after the stress test (p = 0.002) in comparison with the melanoma group (p = 0.407). CCL5/Rantes decreased significantly in the melanoma group, whereas CD16/CD56+ natural killer cells increased in both groups, with a sharp decrease below baseline after stress in the melanoma group (p = 0.001). This pilot study shows an altered immunological response to stressors in melanoma patients.

Measurement of tumor necrosis factor-alpha, interleukin-6, Fas ligand, interleukin-1α, and interleukin-1ß in the aqueous humor of patients with open angle glaucoma using multiplex bead analysis.

PURPOSE: Various cytokines, including tumor necrosis factor-alpha (TNF-α), Fas ligand (FasL), interleukin-1α (IL-1α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), contribute to the pathogenesis of primary open angle glaucoma (POAG). The present study was set to measure these cytokines in the aqueous humor of patients with POAG and in control subjects using multiplex bead analysis. METHODS: Twenty-five patients with POAG and 29 control subjects were enrolled in this case-control study. Aqueous humor concentrations of the cytokines (IL-1 α, IL-1 ß, IL-6, FasL, and TNF- α) were measured using multiplex bead analysis. RESULTS: Mean aqueous humor levels of IL-6 were significantly lower in patients with POAG compared to the control subjects (9.3±23.7 versus 55.3±94.4 pg/ml; p=0.002). No significant difference in the aqueous humor concentration of IL-1ß was found between patients with POAG and control subjects (0.5±0.8 versus 0.4±0.8 pg/ml; p=0.85.) Concentrations of IL-1α, TNF-α, and FasL were below limits of detection. No significant correlation was found between IL-6 concentration and age, duration of disease, cup/disc ratio, or mean deviation. CONCLUSIONS: In the present study, we found significantly lower concentrations of IL-6 in the aqueous humor of patients with POAG.

[Laboratory diagnostics for early detection of rheumatic autoimmune diseases: a guide for the general practitioner]. / Labordiagnostik zur Früherkennung und Verlaufskontrolle von systemischen Autoimmunerkrankungen des rheumatischen Formenkreises in der allgemeinmedizinischen Praxis.

Autoimmune diseases are a clinically heterogeneous group of disorders that represent a challenge for the general practitioner in daily routine. Except for rheumatoid arthritis, which is one of the most frequent autoimmune diseases with a prevalence of approximately 1 % of the population, systemic autoimmune disorders are rare. Thus outside specialized wards it might be a challenge to diagnose the underlying autoimmune disease considering the often kaleidoscopic clinical manifestations. Together with careful anamnesis and suspicious clinical symptoms determination of specific autoantibodies can support the suspected diagnosis. The Austrian group of the European autoimmune standardization initiative (EASI) firstly published this guide 2009 with the aim to provide a map through the jungle of the biomarkers for autoimmune diseases for the general practitioner.

Do children and adolescents with type 1 diabetes mellitus have a higher frequency of parietal cell antibodies than healthy controls?

OBJECTIVES: Parietal cell antibodies (PCA) are markers of autoimmune gastritis (AG). AG can lead to hypergastrinemia and iron-deficiency anaemia (IDA). Compared to healthy controls, adults with type 1 diabetes mellitus (T1DM) show a higher prevalence of PCA (1% vs 20%). The aim of the present study was to evaluate the frequency of PCA in children and adolescents with T1DM compared to healthy controls and the clinical and biochemical markers. PATIENTS AND METHODS: We studied 170 patients (87 boys) with T1DM (mean age 12.9 years) and 101 healthy controls (49 boys; mean age 13.0 years). PCA, free T4, free T3, thyroid-stimulating hormone (TSH), and thyroid antibodies were measured in all of the patients. In addition, gastrin, pepsinogen I, iron, ferritin, vitamin B12, and folate were measured in patients with T1DM only. Gastroscopy was carried out in patients with T1DM having high (>100 U/mL) PCA levels. RESULTS: The frequency of PCA in patients with T1DM was 5.29% compared to 1.98% in healthy controls (not significant). PCA was strongly correlated to both thyroid peroxidase antibodies (TPOAb) and gastrin levels (P = 0.001). IDA was present in 4 of 9 patients from the PCA-positive group compared to 4 of 160 patients from the PCA-negative group. Hypergastrinemia was found in 2 PCA-positive patients. Histopathologically, 1 of 4 patients showed early symptoms of AG. CONCLUSIONS: Children and adolescents with T1DM have a lower frequency of PCA than is reported for adults. Compared to healthy controls, they seem to be at increased risk for developing PCA, in particular if positive for TPOAb, but overt clinical disease is rare in children with T1DM.

Evaluation of Endothelial Dysfunction and Inflammatory Vasculopathy After SARS-CoV-2 Infection-A Cross-Sectional Study.

Background: Rising data suggest that COVID-19 affects vascular endothelium while the underlying mechanisms promoting COVID-19-associated endothelial dysfunction and inflammatory vasculopathy are largely unknown. The aim was to evaluate the contribution of COVID-19 to persisting vascular injury and to identify parameters linked to COVID-19-associated endothelial dysfunction and inflammatory vasculopathy. Methods: In a cross-sectional design, flow-mediated dilation (FMD), nitroglycerine-related dilation (NMD), pulse-wave velocity (PWV), augmentation index, intima-media thickness (IMT), compounds of the arginine and kynurenine metabolism, homocysteine, von Willebrand factor (vWF), endothelial microparticles (EMP), antiendothelial cell antibodies, inflammatory, and immunological parameters, as well as nailfold capillary morphology were measured in post-COVID-19 patients, patients with atherosclerotic cardiovascular diseases (ASCVD) and healthy controls without prior or recent SARS-CoV-2 infection. Results: Post-COVID-19 patients had higher values of PWV, augmentation index, IMT, asymmetric and symmetric dimethylarginine, vWF, homocysteine, CD31+/CD42b- EMP, C-reactive protein, erythrocyte sedimentation rate, interleukin-6, and ß-2-glycoprotein antibodies as well as lower levels of homoarginine and tryptophan compared to healthy controls (all with p < 0.05). A higher total number of pathologically altered inflammatory conditions and higher rates of capillary ramifications, loss, caliber variability, elongations and bushy capillaries with an overall higher microangiopathy evolution score were also observed in post-COVID-19 patients (all with p < 0.05). Most parameters of endothelial dysfunction and inflammation were comparably altered in post-COVID-19 patients and patients with ASCVD, including FMD and NMD. Conclusion: COVID-19 may affect arterial stiffness, capillary morphology, EMP and selected parameters of arginine, kynurenine and homocysteine metabolism as well as of inflammation contributing to COVID-19-associated endothelial dysfunction and inflammatory vasculopathy.

The use and diagnostic value of testing myositis-specific and myositis-associated autoantibodies by line immuno-assay: a retrospective study.

AIMS: Line immune-assays (LIA) for the detection of myositis-specific antibodies (MSA) are used widely for characterization of idiopathic inflammatory myopathies (IIM). Their current use and significance for the diagnosis of IIM remains unclear. METHODS: In this retrospective analysis, we retrieved clinical diagnoses of patients tested for MSA and myositis-associated antibodies (MAA) Jo-1, Mi-2α, Mi-2ß, TIF1γ, SRP, MDA-5, NXP-2, SAE, PL-7, PL-12, EJ, OJ, PM-Scl100, PM-Scl75 and Ku. We calculated clinical specificity, clinical sensitivity, negative- and positive predictive values (PPV) as well as positive and negative likelihood ratios. RESULTS: In total, we analyzed 3167 samples. After exclusion of repeated measurements and patients with insufficient clinical information, data of 1118 patients were available for analysis. A total of 242 patients tested positive for at least one antibody, of which 45 patients had a diagnosis of IIM; 25 IIM patients were negative for all MSA/MAA. Clinical specificity of MSA/MAA for the diagnosis of IIM ranged between 94.2% and 99.9%. Clinical sensitivity and PPV across all antibodies tested ranged from 0.0% to 12.9% and 0.0% to 72.7%, respectively. CONCLUSION: In clinical practice MSA/MAA are used widely for diagnostic work-up of IIM, resulting in a low pre-test probability. Clinicians should be aware that PPVs for most MSA/MAA are low.

Detection of single molecules: solution-phase single-molecule fluorescence correlation spectroscopy as an ultrasensitive, rapid and reliable system for immunological investigation.

More sensitive techniques in molecular and clinical immunology are essential for the development of reproducible profiles. We have developed a novel methodology named solution-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) that fulfils these demands. It is based on the quantification of the probability density of single molecule events in solution is necessary. For example, the Brownian motion of the fluorophore rhodamine-green is detected. Counting about 100,000 photon counts per second and per molecule permits the identification of one single compound. In order to study the applicability of SPSM-FCS in immunology, we have detected and identified a larger nonfluorescent substance in a very complex mixture. The 'unknown' molecules studied were the autoantibodies in serum samples directed against the antigen alpha 3 chain of type IV collagen. In both systems, we were able to characterize the probability density of single fluorescent molecules by means of the averaged absolute molecule number without any calibration. The specific molecules exhibited a Poisson distribution in solution in terms of their 'critical' bulk concentration below about 1 nM. This proof of principle indicates how the SPSM-FCS methodology could be used in immunoassays.

A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 1. Theory and a first application.

Just because there is an average of one molecule in the observation volume of a solution or membrane (single-phase), one cannot say that this is an individual molecule since many different single molecules measured one by one or the same single, individual molecule not leaving the detection volume on time average can cause a single-molecule event. The latter case is of interest and allows the continuous observation of one and the same single molecule without averaging over many 'different' single molecules. For the first time a universal theoretical and experimental framework is presented for the continuous observation of the same single, individual molecule without immobilization, hydrodynamic flow, or burst size histograms of fluorescence intensity traces. In this original article, the stochastic approach is derived and its main characteristics are demonstrated with the free fluorophore rhodamine-green in solution for simpler experimental realization. Single (solution)-phase single-molecule fluorescence auto- (or two-color cross-) correlation spectroscopy (SPSM-FCS) is used as a specific application in order to count the absolute number of molecules in the observation volume. The absolute number of molecules, the diffusion coefficient of the single fluorescent molecule, the lower limit of distance, and the molar concentration of the bulk phase (solution) were directly obtained from the measured auto- or (cross)-correlation curves of the SPSM-FCS experiments. For this purpose, the detection volume that was measured was less then 1 fl (10(-15) l). Then, a concentration of the bulk solution was chosen in such a way that the probability of detecting more than one molecule in the detection volume was very small. The Poisson probability was experimentally determined for the absolute number of molecules depending upon a specified bulk concentration. From the diffusion coefficient of the molecule, it was found that the probability of the molecule diffusing out of the probe volume during the measurements was negligibly small.

A new concept for ultrasensitive fluorescence measurements of molecules in solution and membrane: 2. The individual immune molecule.

In the accompanying original article, the universal theoretical and experimental framework was developed for quantifying one and the same single (selfsame), individual fluorescent-tagged biological molecule without immobilization, hydrodynamic flow or photon burst analysis of fluorescence intensity traces. In the present original article, we describe an application to the detection and identification of circulating anti-glomerular basement membrane antibodies (BMAs) in Goodpasture syndrome. The same single, individual two-color molecule complex was observed among many other molecules. The molecule consisted of the green-tagged antigen, sandwiched autoantibody and red-tagged secondary (detecting) antibody. A 200-fold increase in sensitivity was obtained as compared to the conventional ELISAs on solid phase. This novel concept has several advantages, namely (i) the sensitivity to detect an individual molecule in solution; (ii) the association of the signal with the reaction event, independent of any immobilization procedure and the artifacts thereof; (iii) the assessment of the broad field of natural antibodies. The theoretical and experimental results obtained bring advanced ultrasensitive analytics to the direct investigation of one and the same single, individual immune molecules as exemplified by the experiments performed with Goodpasture antibody. The novel universal theoretical and experimental framework for continuous measuring the same single, individual immune molecule can be readily transferred to other applications.

Clinical applicability of mass spectrometry for inhaled carbon compounds and the characterization of trace element patterns in body fluids.

So far, chemists, molecular biologists and biochemists have reaped the greatest benefits from mass spectrometry (Aebersold et al., 2003). This type of analysis could, however, be useful in many fields. Mass spectrometry is on its way to the doctor's office (Pusch et al., 2003; Földes-Papp et al., 2002; Henry 1999). The article is focused on laser-activated microprobe mass analysis (LAMMA) and inductively coupled argon plasma mass spectrometry (ICP-MS). Potential applications of the two types of mass spectrometry are demonstrated in clinical medicine. It is the first comprehensive review on qualitative characterization of carbonaceous compounds in lung tissue samples in situ and quantitative trace element determination in body fluids.

Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics.

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

Prolonged endurance challenge at moderate altitude: effect on serum eosinophil cationic protein, eosinophil dynamics, and lung function.

BACKGROUND: Eosinophils contain granule proteins such as eosinophil cationic protein (ECP) that have proinflammatory effects on airways. ECP may be released on activation of eosinophils into the plasma and is widely used as a marker of bronchial hyperreactivity and allergic inflammation. Environmental factors as well as intense physical exertion may influence eosinophil-related bronchial hyperreactivity. STUDY OBJECTIVES: To investigate the effect of endurance exercise at moderate altitude on levels of circulating eosinophils, serum ECP, serum osmolality (sOS), and dynamic pulmonary function parameters in healthy mountaineers. SETTING: Alpine field study performed in the Alps of Upper Styria in Austria. Type of exercise: Ascent of a mountain at maximal speed. PARTICIPANTS: Thirty healthy male volunteers from a troop of military mountaineers. RESULTS: Mean ECP concentration increased by 66% at the summit checkpoint (H2) and remained at 63% above baseline (base checkpoint [H0]) after descent (H4), while the blood eosinophil count decreased concomitantly from 250/microL at H0 (preexercise) to 118/microL (53%) at H2 and to 22/microL (81%) at H4. The total serum ECP concentration adjusted to sOS correlated negatively with blood eosinophil count (r = - 0.37; p < 0.0001) and PaO(2) (r = - 0.34; p < 0.001), but positively with the peak expiratory flow (PEF) [r = 0.45; p < 0.0001]. Although sOS correlated with serum ECP at H2 (r = 0.47; p = 0.02) and at 12 h after the start of the experiment (H12) [r = 0.57; p = 0.003], the relationship between total ECP and sOS (r = 0.19; p = 0.034) was less pronounced. FEV(1) in percentage of FVC (%FEV(1)/FVC) [the Tiffenau test], forced expiratory flow rate at 25% of vital capacity, and PEF were significantly higher at H2 than at H0 and H4. %FEV(1)/FVC decreased to 88% (p < 0.01) and 83% (p < 0.001) predicted at H12 and 24 h after start of the experiment, respectively. CONCLUSION: Results provide strong evidence for nonspecific activation of blood eosinophils during prolonged intense aerobic exercise at moderate altitude, modifying both eosinophil dynamics and regulation of ECP release in healthy subjects.

A new dimension for the development of fluorescence-based assays in solution: from physical principles of FCS detection to biological applications.

Ultrasensitive detection methods such as laser-induced fluorescence represent the current state-of-the-art in analytics. Single-molecule detection in solution has received a remarkable amount of attention in the last few years because of its applicability to life sciences. Studies have been performed on the fundamentals of the detection processes themselves and on some biological systems. Fluorescence correlation spectroscopy (FCS) is the link for ultrasensitive multicomponent analysis, showing possibilities for experiments on molecular interactions. Based on the theoretical background of FCS, this article gives full explanation of FCS and an update of highlights in experimental biology and medicine studied by FCS. We focus on a repertoire of diverse immunoglobulin specificities, a ribosome display system, single-molecule DNA sequencing, and a mutant enzyme generated by random mutagenesis of amino acids. We describe the usefulness and the enormous potential of the methodology. Further, this contribution clearly indicates that FCS is a valuable tool for solution-phase single-molecule (SPSM) experiments in immunobiology and medicine. In experiments with the Goodpasture autoantibody, we worked out conditions for the design of experiments on a complex single molecule in solution. The possibility to use SPSM-FCS as a quantitation methodology opens up other important applications beyond the scope of this article. Original results extending the published studies are presented for the rational foundation of SPSM-FCS. In this original contribution, we deal with experimental systems for biology and medicine where the number of molecules in solution is very small. This article is mandatory for gaining confidence in the interpretation of experimental SPSM-FCS results on the selfsame, individual single molecule in solution.

Laser scanning confocal fluorescence microscopy: an overview.

Innovative and important aspects of laser scanning confocal fluorescence imaging (LSCFI) are presented here as a general overview. We have described and discussed the technology of the procedure in some detail. We also report some of our original work with transmembranous uptake of 5S gamma-globulin on living human leukocytes as an example of one specific application of LSCFI. These original data and results are presented, as well as citing other uses and applications, to show the power of LSCFI technique. The article will hopefully be useful for those not familiar with the methodology and utility of laser scanning confocal fluorescence microscopy. Applications of LSCFI are very diverse, and there are new applications of this technology constantly being developed. Interest is growing in LSCFI, particularly in the pharmacologic and therapeutic areas, as demonstrated in this article.

Detection of multiple human herpes viruses by DNA microarray technology.

BACKGROUND: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample. METHODS: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures. RESULTS: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. CONCLUSIONS: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.

C677T single nucleotide polymorphisms of the human methylene tetrahydrofolate reductase and specific identification : a novel strategy using two-color cross-correlation fluorescence spectroscopy.

BACKGROUND: A methylene tetrahydrofolate reductase (MTHFR) deficiency at site C677T renders the enzyme thermolabile and consequently represents a risk factor for vascular disease, neural tube defects, preeclampsia, and thrombosis. Highly specific identification techniques for genotyping are mandatory to give guidance for the diagnosis and monitoring of this deficiency. METHODS: A new approach for performing genotyping has been introduced with the identification of single nucleotide polymorphisms of the human MTHFR. It is based on PCR followed by two-color cross-correlation fluorescence spectroscopy (FCS). Experiments were carried out with green- and red-tagged allele-specific primers, which were fully compatible with the two-color fluorescence cross-correlation setup at 488 nm and 633 nm excitation wavelengths. RESULTS: The measured data of the amplification mixes (tubes) were normalized as the maximum correlation amplitude of each tube. Correlated and uncorrelated data were optically separated in the amplification mixes by their characteristic correlation times, which significantly differed from each other. The correlated data were generated in the presence of the proper mutated genotype template, whereas uncorrelated data were due to the absence of the proper genotype template. Furthermore, the specific association of the two-color fluorescence correlated signals with the target DNA was experimentally proven. Using this novel two-color cross-correlation approach, the MTHFR genotypes, which were determined in 21 clinical samples, showed concordance with methods involving a PCR-based assay with hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxy-fluorescein (FAM)-tagged allele-specific primers and a subsequent separation step with capillary electrophoresis, yet are simpler to perform. There was no evidence of a central trend of false-positive or false-negative results. We demonstrated how the novel, ultrasensitive typing system could be applied to studies where researchers are trying to perfect their assays and are often working with the unknown, or application to problematic assays in a clinical environment for those involved in molecular diagnosis. CONCLUSIONS: We present an alternative method to those commonly used in genotyping. Two-color cross-correlation FCS allows the detection of the fluorescence signals specifically associated with the heterozygous mutated, the homozygous mutated, and normal individuals, as exemplified in this study. The presence of nonspecific amplification products, which interfere with subsequent DNA analysis, could therefore highlight the need for two-color cross-correlation FCS as a means of discriminating between specific association of the fluorescence signals with the target DNA and DNA not related to the target.

Sequential thoracentesis: minor change of zinc compared to other selected essential trace elements in the serum.

This study first indicates that the serum trace element Zn tends to decrease in the course of sequential thoracenteses. Other selected essential elements such as copper (Cu), manganese (Mn), molybdenum (Mo), and cobalt (Co) do not reveal loss changes in their serum levels. Therefore, Zn should be monitored in patients who undergo repeated thoracentesis. To measure the magnitude of changes in serum trace elements and the clinical relevance of potential imbalances, concentrations of the essential elements are analyzed in 57 serum/effusion pairs obtained from 5 patients (4 male, 1 female; age 28-78 yr) who underwent repeated thoracenteses as a result of recurrent pleural effusion. All patients declined other therapeutic options such as chemical pleurodesis and/or chest tube placement. The total volumes of fluid removed ranged from 2.3 to 19.3 L and the frequency of thoracentesis ranged from 6 to 15 within a period of 102-174 days. Two patients had benign pleural disease and three had malignancies. Three patients suffered from pleural effusions resulting from exudates (total protein content > 3.0 g/dL, LDH > 200 U/L), and two resulting from transudates (total protein < 3.0 g/dL, LDH < 200 U/L). All trace elements were simultaneously determined by inductively coupled argon plasma-mass spectrometry. In addition, the concentrations of the following clinically relevant parameters were analyzed by standard methods: total protein, pH, leukocyte count, lactate dehydrogenase, and glucose.

Detection of graphite using laser microprobe mass analysis of a transbronchial biopsy from a foundry worker with mixed dust pneumoconiosis.

Inhalation of dust containing graphite can cause lung disease in foundry workers and workers in graphite mines or mills. Mixed dust pneumoconiosis caused by long-term occupational exposure to graphite dust is a rare disease. Only a few cases of graphite pneumoconiosis have been reported in literature, and these were usually diagnosed post mortem. Our report is of an 80-year-old male patient who had worked in an iron foundry for 20 years and whose work had entailed regular contact with ground graphite and foundry vapors. Chest x-rays revealed both a reticular and nodular pattern in the lung, moderate apical distractions and pleural scarring, all of which were confirmed by high-resolution computed tomography. Bronchoalveolar lavage and transbronchial biopsies were also consistent with mixed dust pneumoconiosis, and due to the long-term dust exposure, graphite pneumoconiosis was strongly suspected. To confirm this diagnosis, the chemical composition of the dark granules in the semi-thin histological sections of the transbronchial biopsies were analyzed using laser microprobe mass spectroscopy. The mass spectra of these black particles were consistent with those of natural graphite powder. Comparative analyses of normal lung tissue did not produce similar spectral patterns. We conclude that histology and cytology does not always suffice to confirm a diagnosis of graphite pneumoconiosis, because black particles are also found in conditions resulting from other exposures, such as heavy smoking or coal mining. Analysis of the composition of particles deposited in the lung tissue offers more precise information, which can be used as evidence in occupational and forensic medicine. Laser microprobe mass spectroscopy can assess the mineral dust load in lung samples.