Recent years brought great focus in the field of development of extracellular vesicles (EVs) based drug-delivery systems. Considering possible applications of EVs as a drug carriers the isolation process is a crucial step. To solve problems related with EV isolation, we created and validated a new EVs isolation method – Low Vacuum Filtration (LVF) and compared it with two commonly applied procedures - differential centrifugation (DC) and ultracentrifugation (UC). EVs isolated from endothelial cells culture media have been characterized by a) transmission electron microscopy (TEM) b) nanoparticle tracking analysis (NTA), c) western blot and d) Fourier-Transform Infrared Spectroscopy (FTIR). Additionally, the membrane surface have been imaged with Environmental Scanning Electron Microscopy (ESEM). We showed that LVF is reproducible and efficient method for EVs isolation form conditioned media. Additionally, we observed correlation between ATR-FTIR spectra quality and the EVs and proteins concentration. ESEM imaging confirmed that actual pore diameter are close to the values calculated theoretically. LVF method is an easy, fast and inexpensive EVs isolation method which allows for isolation of both ectosomes and exosomes from high volume sources with good repeatability. We think that it could be an efficient alternative for commonly applied methods.

Vaccination against HPV significantly reduced the incidence of HPV-related le-sions worldwide. Considering the increasingly younger age of patients in the gynaecological office and the resulting earlier sexual initiation and potentially contact with the HPV virus, doctors need to have tools to verify the diagnosis. Nowadays, women plan motherhood later, so it is necessary to consider whether sexual treatment in the form of, among others, LEEP-conization may increase the risk of premature birth or difficulty dilating the cervix during labour.For this reason, to avoid overtreatment of LSIL changes, methylation testing may be considered. In patients with histopathologically confirmed HSIL during biopsy and ultimately a lower diagnosis - i.e., LSIL or no signs of atypia - methylation turned out to be a useful tool. We performed Pap-smear, HPV genotyping, punch biopsy, LEEP-conization (if needed) and methylation test in 108 women admitted to the District Public Hospital in Poland. Women with a negative methylation test result were significantly more likely to be ultimately diagnosed with LSIL (p=0.013). This means that in 85.7% of patients with HSIL, major cervical surgery could have been avoided if methylation was negative. Methylation testing, as well as dual-staining and diagnostics detecting mRNA transcripts of highly oncogenic types of HPV, might be used in future in the diagnosis of pre-cancerous conditions, mainly of the cervix, and in HPV-dependent cervical cancer screening. The methylation test may also be used in the diagnosis and identification of lesions within the cervical canal, including those located deep within the frontal crypts, not visible even during a professional colposcopic evaluation of the cervix, as well as within the crypts of the palatine tonsils - in diagnostics of head and neck diseases.

Cervical cancer is 4th the most common cancer in women worldwide. Due to the prevalence of HPV in the population (80-90%), the scientists are likely to discover even more associations of this pathogen with other diseases in the future. In recent years, ISH assays that use automated signal-detecting methods in ormalin-fixed, paraffin-embedded (FFPE) cervical tissue, such as the enzyme-categorized signal-detecting system, have shown a higher sensitivity. To evaluate automatic signal detection of in situ hybridization (ISH) assay for detecting human papillomavirus (HPV) DNA, we compared the ability of an ISH probe, Inform HPV II and III (Ventana Medical Systems, Tucson, AZ), to that of PCR assays to detect HPV DNA in cervical tissue specimens with cervical intraepithelial neoplasia (CIN; CIN 1, 28 cases; CIN 2, 22 cases; and CIN 3, 20 cases) and normal cervix (2 cases). Our findings showed significant relation was confirmed between ISH III level and HPV outcome (positive/negative). Patients with positive HPV outcomes had significantly lower ISH III levels, MD = -7961.82 CI95 [-17230.00; -199.21], p = 0.005. Automatic signal detection of ISH assay is not particularly applicable to cervical tissue material. A more useful method of confirming the presence of HPV in the cervix is ​​the HPV test with genotyping, as it allows for collecting a larger amount of material from the cervical disc and canal. The interpretation of a positive or negative ISH test must be guided in the context of clinical history and morphology.