Flow Cytometry as an Alternative to Microscopy for the Differentiation of BAL Fluid Leukocytes.

BACKGROUND: Microscopy is currently the gold standard to differentiate BAL fluid (BALF) leukocytes. However, local expertise for microscopic BALF leukocyte differentiation is often unavailable in clinical practice. RESEARCH QUESTION: Can automated flow cytometry be used instead of microscopy to differentiate BALF leukocytes? STUDY DESIGN AND METHODS: A new automated flow cytometric method for BALF leukocyte differentiation, using four antibodies (anti-CD45, anti-CD66b, anti-HLA-DR, anti-CD52) given to human BALF in one tube, was developed and prospectively validated in 745 unselected subsequent BALF samples from patients with interstitial lung diseases (455 patients), infectious diseases (196 patients), and other diseases (94 patients). Flow cytometry and traditional microscopy were performed by separate investigators in a double-anonymized fashion. Results were compared using Spearman correlation, Deming regression, and Bland-Altman analysis. RESULTS: There was a strong correlation between flow cytometric and microscopic results regarding macrophage/monocyte, lymphocyte, eosinophil, and neutrophil percentages in BALF (P < .001 for all leukocyte subpopulations). Bland-Altman analyses showed that the mean differences between the methods were ≤ 2% for all four cell types. Flow cytometric results differed less than 20% from microscopic results in more than 95% of all samples. Subgroup analyses confirmed that these results were independent from total leukocyte counts in BALF. INTERPRETATION: We report, to our knowledge, the first validated flow cytometric method for BALF leukocyte differentiation, which can be used in clinical settings where local expertise for microscopic analysis is unavailable and which can be combined easily with lymphocyte surface marker analysis.

Response to Various Biologics in Patients with Both Asthma and Chronic Obstructive Pulmonary Disease.

INTRODUCTION: Patients can have features of both chronic obstructive pulmonary disease (COPD) and asthma. However, there is still no consensus how to precisely define this patient population. In addition, there are little data on the effectiveness of biologics in these patients. METHOD: Presence of COPD was defined by a smoking history of ≥10 pack years (PY), a postbronchodilator FEV1/FVC ratio < lower limit of normal (LLN) and FEV1 < 80% predicted, a carbon monoxide diffusion capacity (DLCO) < LLN, and dyspnoea on exertion as a leading symptom. Presence of asthma was defined by high type 2 biomarkers (blood eosinophils ≥300 cells/µL and/or FeNO ≥50 ppb), typical clinical features of asthma (including nocturnal respiratory symptoms), and a documented history of a clinical benefit from inhaled and/or oral glucocorticoid treatment. We analysed data from 20 patients fulfilling the criteria for both COPD and asthma who were newly treated with a biologic due to recurrent exacerbations despite high-dose inhaled triple therapy. RESULTS: Median values before treatment with a biologic were as follows: 40 PY, FEV1 42% predicted, DLCO 45% predicted, 475 eosinophils/µL blood, FeNO 48 ppb. Median duration of biologic treatment (mepolizumab, benralizumab, dupilumab, omalizumab, or tezepelumab) was 12 months. There were significant improvements in exacerbations (most prominent effect), asthma control, and lung function during biologic treatment. CONCLUSIONS: Various types of biologics approved for severe asthma treatment can be effective in patients with both COPD and asthma. We propose an easy-to-use definition of these patients for routine clinical practice.

Cervical spine injuries requiring surgery in a Level I trauma centre in a major German city.

BACKGROUND: Cervical spine injuries (CSI) are rare in trauma patients, at about 9.2-16.5/100,000 inhabitants in Scandinavia and Canada, and the annual incidence of CSI surgeries in Norway is around 3.0/100,000 inhabitants. However, despite their rarity, the incidence of CSI has increased, thereby assuming an increasing need for surgery. Outside of Scandinavia, no data about the incidence of CSI and subsequent surgeries exist. Therefore, this study aimed to analyse CSI epidemiology and surgery in a German city with a Level I trauma centre both to understand the injury and improve needs-based planning. METHODS: This retrospective, monocentre study included all patients who presented with CSI from 2012-2017 at a university hospital with a Level I trauma centre in a major German city and had permanent residency within the city. Based on the assumption that the patients represented all CSI injuries in the city, as they were treated at the only available Level I trauma centre, the annual incidence of surgeries and neurologic deficits due to CSI were calculated. RESULTS: A total of 465 patients with 609 CSI were identified. Of these patients, 61 both received surgery and resided in the city (mean age, 68.1 ± 18.3 years; 26 female, 35 male). The incidence of CSI surgeries was calculated as 3.24/100,000 person years (1.75/100,000 in the upper and 1.54/100,000 in the subaxial cervical spine). Neurologic deficits occurred in 0.64/100,000 person years. The incidence of both surgeries and neurologic deficits showed no significant changes over the 6-year study period. CONCLUSIONS: Compared to Scandinavia, an increasing annual incidence for CSI surgeries and neurologic deficits were found. For long-term demand planning with adaptability to demographic changes, cross-regional studies including long-term follow-up are necessary.

Spatial mapping of VOC exhalation by means of bronchoscopic sampling.

Breath analysis holds promise for non-invasive in vivo monitoring of disease related processes. However, physiological parameters may considerably affect profiles of exhaled volatile organic substances (VOCs). Volatile substances can be released via alveoli, bronchial mucosa or from the upper airways. The aim of this study was the systematic investigation of the influence of different sampling sites in the respiratory tract on VOC concentration profiles by means of a novel experimental setup. After ethical approval, breath samples were collected from 25 patients undergoing bronchoscopy for endobronchial ultrasound or bronchoscopic lung volume reduction from different sites in the airways. All patients had total intravenous anaesthesia under pressure-controlled ventilation. If necessary, respiratory parameters were adjusted to keep PETCO2 = 35-45 mm Hg. 30 ml gas were withdrawn at six sampling sites by means of gastight glass syringes: S1 = Room air, S2 = Inspiration, S3 = Endotracheal tube, S4 = Trachea, S5 = Right B6 segment, S6 = Left B6 segment (S4-S6 through the bronchoscope channel). 10 ml were used for VOC analysis, 20 ml for PCO2 determination. Samples were preconcentrated by solid-phase micro-extraction (SPME) and analysed by gas chromatography-mass spectrometry (GC-MS). PCO2 was determined in a conventional blood gas analyser. Statistically significant differences in substance concentrations for acetone, isoprene, 2-methyl-pentane and n-hexane could be observed between different sampling sites. Increasing substance concentrations were determined for acetone (15.3%), 2-methyl-pentane (11.4%) and n-hexane (19.3%) when passing from distal to proximal sampling sites. In contrast, isoprene concentrations decreased by 9.9% from proximal to more distal sampling sites. Blank bronchoscope measurements did not show any contaminations. Increased substance concentrations in the proximal respiratory tract may be explained through substance excretion from bronchial mucosa while decreased concentrations could result from absorption or reaction processes. Spatial mapping of VOC profiles can provide novel insights into substance specific exhalation kinetics and mechanisms.

Impact of an increase in the inhaled corticosteroid dose on blood eosinophils in asthma.

Here, we report that increasing treatment with inhaled corticosteroids (ICS) in patients with not well-controlled asthma from a medium to a high dose results in a profound reduction of blood eosinophils (median fall in blood eosinophil concentrations from 560 to 320 cells/µL). Therefore, 'normal values' of blood eosinophils in patients with asthma need to be considered in view of the individual ICS doses of the patients. In addition, increases in the dose of ICS may result in blood eosinophil concentrations which would formally preclude treatment with biologics targeting the interleukin-5 pathway.

Correlation of Kondo effect and molecular conformation of the acceptor molecule in the TTF-TCNE charge transfer complex.

A Kondo resonance has been observed on purely organic molecules in several combinations of charge transfer complexes on a metal surface. It has been regarded as a fingerprint of the transfer of one electron from the donor to the extended π orbital of the acceptor's LUMO. Here, we investigate the stoichiometric checkerboard structure of tetrathiafulvalene (TTF) and tetracyanoethylene (TCNE) on a Au(1 1 1) surface using scanning tunneling and atomic force microscopy at 4.8 K. We find a bistable state of the TCNE molecules with distinct structural and electronic properties. The two states represent different conformations of the TCNE within the structure. One of them exhibits a Kondo resonance, whereas the other one does not, despite of both TCNE types being singly charged.

Bronchoalveolar lavage for the diagnosis of Pulmonary Langerhans cell histiocytosis.

BACKGROUND: The histologic diagnosis of Pulmonary Langerhans cell histiocytosis (PLCH) is invasive and can cause complications. To confirm the diagnosis of PLCH, guidelines therefore recommend measuring CD1a-positive bronchoalveolar lavage fluid (BALF) cells despite its poor sensitivity and specificity. Thus, an improved diagnostic accuracy of BALF cell analysis would be desirable. METHODS: Using four-colour flow cytometry, plasmacytoid and myeloid dendritic cells (DCs) were analysed in BALF of 10 newly diagnosed, untreated, smoking patients with PLCH, and compared with BALF DCs from 40 asymptomatic smokers and 21 never-smokers. RESULTS: Compared with controls, myeloid DCs (median: 0.79% of BALF leukocytes) and their subpopulation of Langerhans cells (median: 0.44% of BALF leukocytes) were not increased in PLCH. Patients with PLCH displayed a normal expression of the maturity marker CD83 on BALF myeloid DCs. However, the expression of the co-signaling molecule CD80 on BALF myeloid DCs was significantly lower than in both control groups, with the lowest expression found in more severe disease (presence of cysts > 2 cm in diameter). Based on receiver operating characteristic (ROC) curve analysis, a cut-off of 53% CD80-positive BALF myeloid DCs was optimal for the diagnosis of PLCH, yielding a sensitivity of 0.90 and a specificity of 0.90. CONCLUSIONS: BALF Langerhans cells are not increased in PLCH. However, PLCH is characterised by a low expression of CD80 on BALF myeloid DCs. Due to its considerably higher sensitivity and specificity, this marker appears to be more appropriate to diagnose PLCH than the currently recommended marker CD1a.

Overweight is a predictor of long-term survival in hospitalised patients with exacerbations of COPD.

BACKGROUND: Although hospitalisations due to an exacerbation of chronic obstructive pulmonary disease (COPD) are associated with increased risk of mortality, there is little information on long-term survival after severe COPD exacerbations. METHODS: The 5-year and 8-year overall survival after hospitalisation due to a COPD exacerbation was explored. In addition, potential predictors of survival were analysed. RESULTS: The 57 patients with COPD included in this analysis had a median age of 70 years, a median smoking history of 30 pack years and a median forced expiratory volume in the first second (FEV1) of 41.6% predicted at the time of COPD exacerbation. The majority of the patients had either normal weight (body mass index, BMI 18.5-24.99 kg/m(2): 42%) or overweight (BMI ≥ 25 kg/m(2): 54%). The 5-year overall survival after exacerbation was 54%, the 8-year overall survival 42%. The presence of cardiac comorbidities, a FEV1 <50% predicted, an age >70 years and a BMI <25 kg/m(2), but not smoking history or current smoking, were associated with decreased overall survival. Multivariate regression analysis revealed that only BMI, age and FEV1 were independent predictors of long-term survival. Overweight patients (BMI ≥ 25 kg/m(2)) had a substantially higher 5-year overall survival (74%) than patients with a BMI < 25 kg/m(2) (31%). CONCLUSION: Nearly half of the patients hospitalised due to an exacerbation of COPD die within 5 years after the event. Overweight is a positive predictor of long-term survival in these patients.

Novel strategies for the treatment of asthma.

Novel treatment strategies are currently emerging for patients with inadequately controlled asthma despite good adherence and trigger avoidance. These strategies serve primarily to reduce or completely avoid long-term oral corticosteroid therapy. A number of these options have already been implemented in practice or will soon be authorized for the treatment of asthma, while others still need to prove their clinical practicability, safety and efficacy. The present article provides an overview of the broad spectrum of novel inhaled, oral, systemic, and invasive treatment strategies for asthma.

Nosocomial infection due to Enterococcus cecorum identified by MALDI-TOF MS and Vitek 2 from a blood culture of a septic patient.

We report the case of a nosocomial infection due to Enterococcus cecorum isolated from a blood culture of a 75-year-old septic male patient. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Vitek 2 succeeded in identification of the isolate.

Imbalance of dendritic cell co-stimulation in COPD.

BACKGROUND: Dendritic cells (DCs) control immunity and play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the expression of function-associated surface molecules on circulating DCs in COPD is unknown. METHODS: Four-colour flow cytometry was used to compare blood DC surface molecules of 54 patients with COPD (median age: 59 years; median FEV1: 38% predicted, median CAT score: 24) with two age-matched control groups with normal lung function: 21 current smokers and 21 never-smokers. RESULTS: Concentrations of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) and the mDC/pDC ratio did not differ between the groups. The increased expression of BDCA-1, BDCA-3, CD86 and CCR5 on mDCs in patients with COPD did not significantly differ from smokers with normal lung function. In contrast, COPD was specifically characterised by a decreased expression of the anti-inflammatory co-stimulatory molecule PD-L1 on pDCs and an increased expression of the pro-inflammatory co-stimulatory molecule OX40 ligand (OX40L) on mDCs. These changes were not confined to patients with elevated systemic inflammation markers (leukocytes, c-reactive protein, interleukin-6, fibrinogen). The ratio of OX40L to PD-L1 expression (OX40L/PD-L1 ratio), a quantitative measure of imbalanced DC co-stimulation, correlated with the severity of pulmonary emphysema in patients with COPD. CONCLUSION: An imbalance of DC co-stimulation might contribute to the pathogenesis of COPD.