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Elena Arellano-Orden,1,2 Carmen Calero,1,2 Cecilia López-Ramírez,1,2 Verónica Sánchez-López,1,2 José Luis López-Villalobos,1,2 María Abad Arranz,1,2 Ana Blanco-Orozco,1 Remedios Otero-Candelera,1,2 José Luis López-Campos1,21Unidad Médico-Quirúrgica de Enfermedades Respiratorias, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Seville, Spain; 2CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, SpainBackground: Previous studies have shown that the arterial wall is a potential source of inflammatory markers in COPD. Here, we sought to compare the expression of acute phase reactants (APRs) in COPD patients and controls both at the local (pulmonary arteries and lung parenchyma) and systemic (peripheral blood leukocytes and plasma) compartments.Methods: Consecutive patients undergoing elective surgery for suspected primary lung cancer were eligible for the study. Patients were categorized either as COPD or control group based on the spirometry results. Pulmonary arteries and lung parenchyma sections, peripheral blood leukocytes, and plasma samples were obtained from all participants. Gene expression levels of C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2, and SAA4) were evaluated in tissue samples and peripheral blood leukocytes by reverse transciption-PCR. Plasma CRP and SAA protein levels were measured by enzyme-linked immunosorbent assays. Proteins were evaluated in paraffin-embedded lung tissues by immunohistochemistry.Results: A total of 40 patients with COPD and 62 controls were enrolled. We did not find significant differences in the gene expression between COPD and control group. Both CRP and SAA were overexpressed in the lung parenchyma compared with pulmonary arteries and peripheral blood leukocytes. The expression of SAA was significantly higher in the lung parenchyma than in the pulmonary artery (2-fold higher for SAA1 and SAA4, P=0.015 and P

Essentials Platelet extracellular vesicles (EVs) concentrations measured by flow cytometers are incomparable. A model is applied to convert ambiguous scatter units to EV diameter in nanometer. Most included flow cytometers lack the sensitivity to detect EVs of 600 nm and smaller. The model outperforms polystyrene beads for comparability of platelet EV concentrations. Summary: Background Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights into the diameter of detected EVs. Objectives To evaluate gates based on the estimated diameter of EVs instead of beads. Methods A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates in order to measure the concentration of CD61–phycoerythrin-positive platelet EVs. Results Of the 46 evaluated FCMs, 21 FCMs detected the 600–1200-nm EV diameter gate. The 1200–3000-nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL min−1 differed six-fold in measured flow rates between instruments. Conclusions EV diameter gates improve the interlaboratory variability as compared with previous approaches. Of the evaluated FCMs, 24% could not detect 400-nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity.

Microparticles (MPs) are potential noninvasive biomarkers for diagnosis or prognosis in pathologic conditions. However, the lack of standardization of the preanalytical and analytical methods leads to a wide variability in MPs results. The recently developed Megamix-Plus beads, a new bead-based standardization tool optimized to specific types of flow cytometers, could help circumvent this problem. The aim of the present study was to determine whether the number of total MPs and platelet-derived MPs (PMPs) is similar using 2 different cytometer platforms calibrated with the Megamix-Plus beads. Blood samples from 65 patients with deep venous thrombosis were collected and processed to obtain platelet poor plasma (PPP). The number of total MPs and PMPs in each PPP sample was measured using 2 flow cytometers. Megamix-Plus side scatter channel beads were used to calibrate the LSRFortessa flow cytometer from Becton Dickinson, whereas Megamix-Plus forward scatter channel beads were applied to the Navios flow cytometer from Beckman Coulter. High correlation of total MPs and PMPs values between the flow cytometers was found (r = 0.908, P < 0.01 and r = 0.910, P < 0.001, respectively). However, the absolute numbers of total MPs and PMPs were significantly higher measured with the Navios flow cytometer compared with the LSRFortessa cytometer. Therefore, both platforms are valid for MPs determination in general, although a similar platform with the same calibration tool could be a better choice for multicenter studies.