Application of a Micro Free-Flow Electrophoresis 3D Printed Lab-on-a-Chip for Micro-Nanoparticles Analysis

The present work describes a novel microfluidic free-flow electrophoresis device developed by applying three-dimensional (3D) printing technology to rapid prototype a low-cost chip for micro- and nanoparticle collection and analysis. Accurate reproducibility of the device design and the integration of the inlet and outlet ports with the proper tube interconnection was achieved by the additive manufacturing process. Test prints were performed to compare the glossy and the matte type of surface finish. Analyzing the surface topography of the 3D printed device, we demonstrated how the best reproducibility was obtained with the glossy device showing a 5% accuracy. The performance of the device was demonstrated by a free-flow zone electrophoresis application on micro- and nanoparticles with different dimensions, charge surfaces and fluorescent dyes by applying different separation voltages up to 55 V. Dynamic light scattering (DLS) measurements and ultraviolet−visible spectroscopy (UV−Vis) analysis were performed on particles collected at the outlets. The percentage of particles observed at each outlet was determined in order to demonstrate the capability of the micro free-flow electrophoresis (μFFE) device to work properly in dependence of the applied electric field. In conclusion, we rapid prototyped a microfluidic device by 3D printing, which ensured micro- and nanoparticle deviation and concentration in a reduced operation volume and hence suitable for biomedical as well as pharmaceutical applications

PDMS-Based Microdevices for the Capture of MicroRNA Biomarkers

The isolation and analysis of circulating biomarkers, the main concern of liquid biopsy, could greatly benefit from microfluidics. Microfluidics has indeed the huge potentiality to bring liquid biopsy into the clinical practice. Here, two polydimethylsiloxane (PDMS)-based microdevices are presented as valid tools for capturing microRNAs biomarkers from clinically-relevant samples. After an extensive study of functionalized polydimethylsiloxane (PDMS) properties in adsorbing/eluting microRNAs, the best conditions were transferred to the microdevices, which were thoroughly characterized. The channels morphology and chemical composition were measured, and parameters for the automation of measures were setup. The best working conditions were then used with microdevices, which were proven to capture microRNAs on all channel surfaces. Finally, microfluidic devices were successfully validated via real-time PCR for the detection of a pool of microRNAs related to non-small cell lung cancer, selected as proof-of-principle. The microfluidic approach described here will allow a step forward towards the realization of an ecient microdevice, possibly automated and integrated into a microfluidic lab-on-a-chip with high analytical potentialities

Prescription appropriateness of anti-diabetes drugs in elderly patients hospitalized in a clinical setting: evidence from the REPOSI Register

Diabetes is an increasing global health burden with the highest prevalence (24.0%) observed in elderly people. Older diabetic adults have a greater risk of hospitalization and several geriatric syndromes than older nondiabetic adults. For these conditions, special care is required in prescribing therapies including anti- diabetes drugs. Aim of this study was to evaluate the appropriateness and the adherence to safety recommendations in the prescriptions of glucose-lowering drugs in hospitalized elderly patients with diabetes. Data for this cross-sectional study were obtained from the REgistro POliterapie-Società Italiana Medicina Interna (REPOSI) that collected clinical information on patients aged ≥ 65 years acutely admitted to Italian internal medicine and geriatric non-intensive care units (ICU) from 2010 up to 2019. Prescription appropriateness was assessed according to the 2019 AGS Beers Criteria and anti-diabetes drug data sheets.Among 5349 patients, 1624 (30.3%) had diagnosis of type 2 diabetes. At admission, 37.7% of diabetic patients received treatment with metformin, 37.3% insulin therapy, 16.4% sulfonylureas, and 11.4% glinides. Surprisingly, only 3.1% of diabetic patients were treated with new classes of anti- diabetes drugs. According to prescription criteria, at admission 15.4% of patients treated with metformin and 2.6% with sulfonylureas received inappropriately these treatments. At discharge, the inappropriateness of metformin therapy decreased (10.2%, P < 0.0001). According to Beers criteria, the inappropriate prescriptions of sulfonylureas raised to 29% both at admission and at discharge. This study shows a poor adherence to current guidelines on diabetes management in hospitalized elderly people with a high prevalence of inappropriate use of sulfonylureas according to the Beers criteria

Application of a Micro Free-Flow Electrophoresis 3D Printed Lab-on-a-Chip for Micro-Nanoparticles Analysis

The present work describes a novel microfluidic free-flow electrophoresis device developed by applying three-dimensional (3D) printing technology to rapid prototype a low-cost chip for micro- and nanoparticle collection and analysis. Accurate reproducibility of the device design and the integration of the inlet and outlet ports with the proper tube interconnection was achieved by the additive manufacturing process. Test prints were performed to compare the glossy and the matte type of surface finish. Analyzing the surface topography of the 3D printed device, we demonstrated how the best reproducibility was obtained with the glossy device showing a 5% accuracy. The performance of the device was demonstrated by a free-flow zone electrophoresis application on micro- and nanoparticles with different dimensions, charge surfaces and fluorescent dyes by applying different separation voltages up to 55 V. Dynamic light scattering (DLS) measurements and ultraviolet−visible spectroscopy (UV−Vis) analysis were performed on particles collected at the outlets. The percentage of particles observed at each outlet was determined in order to demonstrate the capability of the micro free-flow electrophoresis (µFFE) device to work properly in dependence of the applied electric field. In conclusion, we rapid prototyped a microfluidic device by 3D printing, which ensured micro- and nanoparticle deviation and concentration in a reduced operation volume and hence suitable for biomedical as well as pharmaceutical applications

Prevalence of Human Papillomavirus (HPV) and Other Sexually Transmitted Infections (STIs) among Italian Women Referred for a Colposcopy

Sexually transmitted infections (STIs) represent a major cause of morbidity in women and men worldwide. Human Papillomavirus (HPV) infections are among the most prevalent STIs and persistent infections with high-risk HPV (hrHPV) genotypes can cause cervical dysplasia and invasive cervical cancer. The association of other STIs with HPV cervical infection and/or dysplasia has however not yet been fully elucidated. The aim of this study was to assess the prevalence of HPV and other STIs among women presenting with an abnormal cervical cytology. Cervical infections with 28 HPV genotypes and seven other sexually transmitted pathogens were evaluated in 177 women referred for a colposcopy after an abnormal Pap smear. Positivity for at least one hrHPV genotype was shown in 87% of women; HPV 16 was the most prevalent (25.0%), followed by HPV 31 and HPV 51. The overall positivity for other STIs was 49.2%, with Ureaplasma parvum being the most prevalent microrganism (39.0%). Co-infections between hrHPV and other STIs were demonstrated in 17.5% of women; no significant association was demonstrated between multiple infections and the colposcopy findings. This study provides new epidemiological data on the prevalence of cervical infections associated with HPV and seven other common sexually transmitted pathogens in a population of women presenting with an abnormal cervical cytology

Human papillomavirus DNA detection in plasma and cervical samples of women with a recent history of low grade or precancerous cervical dysplasia.

Circulating HPV DNA has been previously described in women with advanced stages of cervical cancer and has been suggested to be a prognostic marker of disease recurrences and metastases. Only a few studies have reported the presence of HPV DNA in bloodstream of patients with low grade or precancerous cervical lesions. This study aimed to define if HPV DNA could be detected in plasma samples of 120 women referred for a recent history of cervical dysplasia who presented with lesions ranging from High Squamous Intraepithelial Lesion (H-SIL) to regressed normal cytology. HPV DNA detection was carried out in both plasma and cervical samples using type-specific real-time quantitative PCR assays identifying oncogenic HPV 16, 18, 31, 33, 45, 51 and 52. Overall, 34.2% (41/120) of plasma samples were shown to be positive for HPV DNA detection; HPV 45 (46.3%), HPV-51 (29.6%), and HPV 16 (18.5%) were the most frequently identified genotypes. The rate of HPV detection in paired cervical and plasma samples increased with advancing disease stage, ranging from 15.4% in women with regressed lesions to 38.9% in women with HSIL; HPV 16 resulted the most common genotype identified in women found to be HPV DNA positive in both cervical and plasma samples. Moreover, HPV 16 showed the highest median viral load value in both cervical and plasma samples, with 48,313 copies/104 cells and 1,099 copies/ml, respectively. Results obtained in this study confirm that HPV DNA can be detected and quantified in plasma samples of women with asymptomatic cervical infection. Further knowledge on HPV dissemination through the blood stream of women with cervical lesions would be very important in better understanding the natural history of HPV infection as well as its potential role in other distant tumors

Analysis of Human Papillomavirus (HPV) 16 Variants Associated with Cervical Infection in Italian Women

This study aims to evaluate HPV16 variants distribution in a population of Italian women living in two different regions (Lombardy and Sardinia) by sequence analyses of HPV16-positive cervical samples, in order to reconstruct the phylogenetic relationship among variants to identify the currently circulating lineages. Analyses were conducted starting from DNA isolated from 67 HPV16-positive cervical samples collected from two different Italian centres (31 from Lombardy and 36 from Sardinia) of women with normal and abnormal cervical cytology. The entire long control region (LCR) and 300 nt of the E6 gene was sequenced to identify intra-type variants. Sequence comparison and phylogenetic analysis were made using a distance-based neighbour joining method (NJ) and Kimura two-parameter model. Data obtained reported that Italian sequences mainly belonged to the European lineage, in particular sublineage A2. Only five sequences clustered in non-European branches: two in North American lineage (sublineage D1), two in African-1 (sublineage B1) and one in African-2. A new 27 nucleotide duplication in the central segment of the LCR region was found in a sequence obtained from a sample isolated in Sardinia. A predominance of European variants was detected, with some degree of variability among the studied HPV16 strains. This study contributes to the implementation of data regarding the molecular epidemiology of HPV16 variants

PDMS-Based Microdevices for the Capture of MicroRNA Biomarkers

none9The isolation and analysis of circulating biomarkers, the main concern of liquid biopsy, could greatly benefit from microfluidics. Microfluidics has indeed the huge potentiality to bring liquid biopsy into the clinical practice. Here, two polydimethylsiloxane (PDMS)-based microdevices are presented as valid tools for capturing microRNAs biomarkers from clinically-relevant samples. After an extensive study of functionalized polydimethylsiloxane (PDMS) properties in adsorbing/eluting microRNAs, the best conditions were transferred to the microdevices, which were thoroughly characterized. The channels morphology and chemical composition were measured, and parameters for the automation of measures were setup. The best working conditions were then used with microdevices, which were proven to capture microRNAs on all channel surfaces. Finally, microfluidic devices were successfully validated via real-time PCR for the detection of a pool of microRNAs related to non-small cell lung cancer, selected as proof-of-principle. The microfluidic approach described here will allow a step forward towards the realization of an efficient microdevice, possibly automated and integrated into a microfluidic lab-on-a-chip with high analytical potentialities.noneLunelli, Lorenzo; Barbaresco, Federica; Scordo, Giorgio; Potrich, Cristina; Vanzetti, Lia; Marasso, Simone Luigi; Cocuzza, Matteo; Pirri, Candido Fabrizio; Pederzolli, CeciliaLunelli, Lorenzo; Barbaresco, Federica; Scordo, Giorgio; Potrich, Cristina; Vanzetti, Lia; Marasso, Simone Luigi; Cocuzza, Matteo; Pirri, Candido Fabrizio; Pederzolli, Cecili

Accuracy of Human Papillomavirus (HPV) Testing on Urine and Vaginal Self-Samples Compared to Clinician-Collected Cervical Sample in Women Referred to Colposcopy

In the context of cervical cancer prevention, where human papillomavirus (HPV) infection is pivotal, HPV testing is replacing Pap Smear in primary screening. This transition offers an opportunity for integrating self-sampling to enhance coverage. We evaluated the accuracy of HPV testing using self-collected urine and vaginal samples, comparing them to physician-collected cervical swabs. From a cohort of 245 women with abnormal cytology, we collected self-sampled vaginal, urine, and clinician-administered cervical specimens. Employing Anyplex™II HPV28 assay, outcomes revealed HPV positivity rates of 75.1% (cervical), 78.4% (vaginal), and 77.1% (urine). Significant, hr-HPV detection concordance was observed between self-taken cervical samples and clinical counterparts (k = 0.898 for vaginal; k = 0.715 for urine). This study extends beyond accuracy, highlighting self-collected sample efficacy in detecting high-grade cervical lesions. The insight underscores self-sampling’s role in bolstering participation and aligns with WHO’s goal to eliminate cervical cancer by 2030