Surgical resection or radiotherapy (RT) are standard approaches for early-staged hypopharyngeal squamous cell carcinoma (HPSCC). Transoral laser microsurgery (TOLMS) seems to provide good oncological and functional results with few local complications. The aim of our study was to analyze the outcomes of TOLMS, with or without neck dissection or RT, in the treatment of HPSCC in a tertiary referral center.A retrospective study was conducted in patients with early T-category (T1–T2) HPSCC treated by TOLMS.A total of 34 patients were included in the study. The series includes 17 (50%) T1 and 17 (50%) T2 classified tumors. The 5-year overall survival and disease-specific survival rates were 51% and 66%, respectively, with a 5-year local control rate of 92%. All patients reassumed oral diet and none of them had a tracheostomy at the end of the follow-up.TOLMS offers an effective treatment option in terms of oncologic control and function preservation in locally circumscribed HPSCC. Hypopharyngeal squamous cell carcinoma (HPSCC) constitutes 3–7% of all head and neck cancers and, despite advancements in surgery and chemo-radiotherapy (CRT), its prognosis remains poor, with one of the highest associated morbidity and mortality of any head and neck squamous cell carcinomas –3.Most patients with HPSCC 70–85%) present with advanced-stage disease (stages III and IV), as a result of the local extension of the tumor and the high incidence of lymph node involvement at the time of diagnosis . 50–70% % presentWithin this context, the aim of the treatment is to optimize survival and provide functional organ preservation, when feasible. Surgical resection or radiotherapy (RT) have become the standard approaches for early-staged tumors (stages I–II). On the other hand, the treatment for patients with locally advanced HPSCC (T3-T4) consists of a total laryngectomy with partial or total pharyngectomy or protocols based on CRT , 6.2 transoral laser microsurgery (TOLMS) and transoral robotic surgery (TORS), as well as partial open procedures [RT (in early stages) or CRT (in advanced stages) does not jeopardize oncological outcomes and allows for satisfactory functional results, thus becoming the treatment of choice in most cases. However, avoiding surgery is not a guarantee of functional preservation, given the early and late side effects of RT/CRT . Althougocedures .TOLMS as treatment for T1-T2 HPSCC has achieved excellent outcomes, such as those obtained with open surgical procedures and RT, with lower postoperative morbidity and optimal functional results , 8, 9. AIn the attempt to provide information on the role of TOLMS in the treatment of HPSCC, the aim of our study was to evaluate the oncologic and functional outcomes of TOLMS for HPSCC at our Department in a tertiary referral center, and to determine the influence of the clinic-pathologic factors in the prognosis of these patients.We reviewed the clinical records of the patients with HPSCC treated with TOLMS at our Department from 2000 to 2018. Written informed consent was obtained from each patient. Data were extracted from the original medical records, and they were retrospectively reviewed and analyzed anonymously, without any further inquiry or patient intervention, following institutional review board guidelines.The variables included were age, sex, tumor location, tobacco and alcohol consumption, familiar and personal history of cancer, stage, histological grade, post-operative complications, and the incidence of recurrence and second primary malignancies. Tumors were classified according the TNM classification of the International Union Against Cancer . In terms of the magnitude of consumption, among the smokers the cumulative doses were calculated according to the pack-year index and among the drinkers according to the grams of ethanol consumed per day. The minimum follow-up of the patients included in the study was 24 months.2 LASER System and AcuPulse CO2 LASER system. Endoscopic resection was performed using en bloc or piecemeal techniques according to several variables, including size and location of the tumor, as well as the tumor exposure. All the patients underwent a transoral laser CO2 microsurgery with curative intent, following Steiner´s recommendations [The surgical procedures were performed under general anesthesia after orotracheal intubation, with Sharplan 30C COndations . The extndations .After being assessed in a multi-disciplinary committee, TOLMS was indicated for all those patients with T1-T2 hypopharyngeal tumors once adequate exposure of the entire lesion was confirmed by microlaryngoscopy. Adequate exposure implies not only a complete view of the lesion, but also sufficient space to allow maneuvering the surgical instruments. In cases where complete resection of the tumor was deemed not possible transorally, a non-surgical treatment was chosen.All patients were assessed by expert anesthesiologists to ensure a preoperative morbidity status good enough to withstand the procedure (ASA 1 or 2).Ipsilateral neck dissection was carried out in patients with lateralized not crossing the midline tumors and no clinical disease on both sides of the neck. The indications for performing a selective neck dissection (levels II–IV) were absence of identifiable metastatic neck nodes or radiological findings of positive nodes without extracapsular spread. The remaining cases received a modified radical neck dissection.During the neck dissection, the ipsilateral superior thyroid artery was prophylactically ligated to reduce the risk of massive hemorrhage. When a neck dissection was not included in the treatment plan, no additional measures were taken to reduce the risk of bleeding.Patients with affected surgical margins, more than 2 positive nodes or extra-nodal extension in the pathological examination, received postoperative intensity-modulated RT within 3 months after surgery, following our hospital´s treatment protocols, which are based on the recommendations of the NCCN guidelines in use at the time. For tumors amenable to larynx-preserving surgery, these guidelines recommend either definitive RT or partial laryngopharyngectomy associated with ipsilateral or bilateral neck dissection, the latter being our treatment of choice. In case of adverse postoperative features, such as positive margins and/or extranodal extension, the NCCN guidelines contemplates RT as an acceptable adjuvant treatment.The follow-up consisted of standard fiberoptic laryngoscopy every 3–6 months. A CT including neck and thorax was performed annually on all patients for the first 5 years of follow-up.The endpoints assessed were OS, disease-specific survival (DSS) and the local control rate (LCR). For DSS, only death from pharyngeal cancer was seen as an event. Survival curves were drawn up according to the Kaplan–Meier product limit estimate. Differences between survival times were analyzed by the log-rank method. In evaluating recurrence, events were defined as local and regional recurrences, distant metastasis and combinations of them.Functional outcomes were assessed based on the need for permanent tracheostomy or enteral feeding tube.p < 0.05 was considered statistically significant for all comparisons.All statistical analyses were performed using IBM SPSS statistics software version 22 for MacOS. The characteristics of the patients and their tumors are summarized in Table Most tumors were localized in the piriform sinus (91%), followed by the postcricoid area (6%) and the posterior wall (3%). Primary tumors were equally distributed in T1 and T2 classifications. However, given the high proportion of patients with nodal metastases, 65% of patients presented an advanced tumor stage (III and IVA). Most tumors were poorly differentiated carcinomas (42%).Surgical margins were microscopically involved in 4 out of 34 cases (12%). All cases with involved margins were T2 tumors.31 patients (91%) underwent a neck dissection, which was performed simultaneously in 26 cases (84%) and staged in 5 (16%). A unilateral neck dissection was carried out in 23 patients (74%) and 8 (26%) underwent bilateral neck dissection. Except in 7 cases, where type II modified radical neck dissection was performed, a selective neck dissection (levels II–IV) was done. From the 31 patients that received a neck dissection, 19 (61%) presented pathological neck metastasis. 3 patients did not undergo neck dissection. All patients had a clinically negative neck and had a staged elective neck dissection planned, but declined it after surgery for the primary tumor.Excluding two cases that died in the postoperative period, all patients with pathological neck metastasis (59%) received adjuvant RT (in one patient with concomitant chemotherapy).Patients had a median postoperative hospital stay of 14 days (range 5–41 days). The major complications were: bleeding in 4 patients (11.7%), respiratory infection in 1 patient (3%) and airway obstruction requiring emergency tracheostomy in 2 cases (6%).2 patients (6%) died in the postoperative period from massive hemorrhage. No differences were identified in either clinical or management aspects in these two patients with respect to the rest of the sample. Both patients were male, aged 59 and 73 years. One of them had a T1 tumor and did not receive a neck dissection, and the other had a T2 tumor and received a simultaneous neck dissection.Among minor complications, 1 patient presented a chylous fistula, resolved with conservative treatment.Excluding the two patients who died in the postoperative period, the mean follow-up time was 46 months (range 24–120 months). At the time of the last follow-up visit, 15 patients (47%) were still alive and free of disease, 8 (25%) had died due to intercurrent disease without evidence of recurrence and 9 (28%) had died with cancer.Recurrent disease  developed in 10 patients (31%): 1 patient (3%) developed local recurrence and 5 of them (16%) had a regional recurrence. One patient (3%) developed both local and regional recurrences. Distant metastases were found in 3 patients (9%), all of them located in the lungs. Nine patients (28%) developed a secondary primary tumor: 5 (55%) of them located in the upper aerodigestive tract area , while 4 of them (45%) were in the lungs. The patient with local recurrence was treated with total laryngectomy and adjuvant RT and subsequently developed lung metastases. The 3 patients with regional recurrence that did not receive postoperative RT were treated with salvage radical neck dissection and adjuvant RT; two of them had additional recurrences and received palliative treatment. The patient with both local and regional recurrence was considered unresectable and received palliative treatment with chemotherapy, as the patients with regional recurrences that received adjuvant RT and the patients with distant metastases.No statistically significant differences were found between the presence of loco-regional recurrence and the T and N classifications, nor with the histological grade or the margin status. However, recurrences were higher in patients that did not receive adjuvant treatment, reaching near statistically significant differences Table . In contThe 5-year OS Fig.  and DSS A nasogastric feeding tube was needed in 26 patients (76%) in the postoperative period. The remaining patients reassumed oral diet the day following surgery. The mean time to achieve oral feeding was 7.4 days (median 6 days) and the mean duration of nasogastric tube feeding was 7.2 days (median 5 days). Nasogastric tube could be removed in 25 patients and 1 patient (3%) required a temporary gastrostomy, that could be removed 6 months later. Two patients required a tracheostomy, that could be also removed, and therefore, none of the patients had a gastrostomy or tracheostomy at the end of the follow-up. Only one patient required a total laryngectomy , so the 5-year laryngectomy-free survival rate was 97%.Patients with primary HPSCC classified T1 or T2, without nodal or distant metastases may be treated with transoral approaches, open partial laryngectomy, or RT. Local surgical treatment is usually accompanied by neck dissection and adjuvant RT when indicated. The lack of prospective randomized trials makes it difficult to compare these approaches. In addition, the different stages, end points, observation periods, statistics and indication criteria of the different treatment modalities make it difficult to compare the efficacy of one treatment over another. In this study we assessed the oncologic and functional results of TOLMS for primary management of HPSCC.Even though TOLMS offers a surgical option in these tumors, because all regions of the hypopharynx are accessible with this approach , studiesWe obtained an excellent LCR of 92% at 5 years, superior to the rates described in the other studies Table  and the p = 0.151, and 83% vs 71%, p = 0.320, respectively). However, the TOLMS had higher laryngeal preservation rates  and better functional outcomes. Although these results are based mostly on small case series, they suggest that TOLMS offers equal, if not better, oncological results than open approaches in the treatment of HPSCC. In addition, among the advantages of TOLMS when compared with open approaches, the main one could be the early recovery of pharyngolaryngeal functions. In most patients, tracheotomy is not necessary and healthy tissue may be maximally preserved to reduce morbidity, as reported by Suarez et al. [Although the LCR obtained with TOLMS is adequate, OS and DSS are compromised due to the high incidence of nodal metastasis (which results in more than 2/3 of patients being in advanced stages) and the frequent development of distant metastasis. Then, the described 5-year OS and DSS are between 37–77% and 58–88%, respectively Table . These op = 0.02). Rabbani et al. [Primary RT/CRT might also be a treatment option for HPSCC that would be surgically resectable. Modern techniques and protocols follow the goal of best tumor control, such as maximal sparing of healthy tissue to diminish toxicity. In early-stage disease, primary RT achieves good oncologic outcome and is an alternative treatment strategy. Nakamura et al.  reportedi et al.  reportedi et al.  reportedi et al.  reportedKeeping in mind that early tumors usually have better prognosis, the goals of treatment should be not only to cure, but also to maintain adequate swallowing and voice function. According to Alvarez-Marcos et al. results, patients with tumors of the larynx and hypopharynx treated with CRT have frequent asymptomatic swallowing disorders that alter their quality of life . It is, The organ preservation rate in our series was 97%, with only one patient requiring a total laryngectomy due to tumor recurrence. This high laryngeal preservation rate was also described in the systematic review of Lane et al. which report laryngeal preservation rates from 89 to 100% (median 97%) . These rThese results, compare well with the laryngeal preservation rates obtained with RT/CRT, with reported rates between 56 and 87% , 25, 26.As observed in our series, adjuvant treatment can be avoided in a large proportion of patients (41%), which may explain the excellent functional results without compromising oncologic outcomes. The high incidence of second primary tumors in this kind of patients represents another reason to avoid unnecessary adjuvant treatments and preserve the option of RT/QRT for such scenarios.However, although no significant differences were identified, a trend toward higher incidence of recurrence was observed in patients who did not receive adjuvant treatment Table . FurtherThe restriction to obtain wide resection margins is an inherent limitation of this surgical technique compared to open approaches and TORS . We wereOne advantage of TOLMS is the low complication rate. In our study, the most common complications were postoperative bleeding that occurred in 11.7% of patients. This is in line with the results of another study investigating TOLMS for HPSCC . AlthougTOLMS offers an effective treatment option in terms of oncologic control and function preservation for locally circumscribed (T1-2) HPSCC, with similar oncological results to open approaches or CRT and improved functional outcomes.


However, extra HL significantly decreased nanoleakage values when applied to eroded and sound dentin (p = 0.0001). After two years, the application of extra HL produced significantly higher microtensile bond strength and lower nanoleakage values than the control group for both adhesives (p = 0.0001). In all cases, sound dentin showed higher microtensile bond strength and lower nanoleakage values than eroded dentin (p = 0.000001). An extra HL increased the bond strength and reduced nanoleakage in eroded dentin after two-years of storage.This paper evaluates the effect of an additional hydrophobic resin coat (extra HL) associated with universal adhesives on sound and eroded dentin and evaluated immediately or after 2 years of water storage to improve the microtensile bond strength (μTBS) and nanoleakage (NL) when compared to the use of universal adhesives only. Sixty-four molars were assigned to eight groups using the following combinations: 1. dentin substrate, including sound and eroded dentin; 2. treatment, including the control and extra HL and storage time (immediately and after two-years of storage). Two universal adhesives  were evaluated. Before restoration, half of the teeth were subjected to soft-drink erosion. Composite buildups were bonded; specimens were stored (37 °C/24 h), sectioned into resin–dentin bonded sticks and tested for microtensile bond strength and nanoleakage using SEM (immediately and after two-years of storage). Three-way ANOVA and Tukey’s test (α = 0.05%) were used. In the immediate testing, the application of extra HL did not increase microtensile bond strength values compared with the control group in either substrate ( The bonding performance and predictability of adhesives are challenging for eroded dentin surfaces ,2,3,4. CPhysical barriers of exposed collagen fibrils (organic matrix) induced by continuous and progressive mineral loss on the eroded surface hinder adequate adhesive infiltration ,2,5. ColWith the continuous loss of tissue, the structural and aesthetic integrity of the teeth may be compromised, resulting in functional and aesthetic problems that require restorative intervention ,10. ThusSeveral clinical alternatives used for sound dentin  have notSeveral researchers have advocated the use of an additional hydrophobic resin coat to improve the bonding performance of adhesives ,25,26,27Therefore, this in vitro study aims to evaluate the effect of an additional hydrophobic resin coat to improve the microtensile bond strength  and decrease the nanoleakage  on eroded dentin bonding, when compared to sound dentin, after two years of water storage in comparison with immediate time.The null hypotheses tested were as follows: (1) the use of an additional hydrophobic resin coat associated to universal adhesives would not affect microtensile bond strength (μTBS) or nanoleakage (NL) values when compared to only universal adhesive application; (2) these microtensile bond strength and nanoleakage values would not result in differences when adhesives would be evaluated on sound vs. eroded dentin and; (3) aging (immediate or after 2 years of water storage) would not affect microtensile bond strength or nanoleakage values.Sixty-four human molars were considered in this study. The teeth were collected after approval from the local ethics committee (#4.310.655). They were disinfected with 0.5% chloramine and stored in distilled water until use. The occlusal third of the crown was removed from all teeth using a diamond saw in a cutter machine with water-cooling  to obtain a flat dentin surface. To confirm the absence of enamel on the dentin surface, careful examination was performed under a stereomicroscope  at 30× magnification. The exposed dentin surfaces were polished with wet #600-grit silicon carbide abrasive paper (SiC) for 30 s to standardize the smear layer.n = 8 dentin specimens) using a combination of the following variables: 1. dentin substrate, including sound and eroded dentin; 2. treatment, including the control (adhesives applied according to manufacturer recommendations) and extra HL  and 3. storage time (24 h and after 2 years). Two universal adhesives  or Scotchbond Universal  were used and the specimens obtained for each tooth were randomly divided and tested . Product information and application mode details for the experimental groups are provided in The teeth were randomly divided into 8 groups  mean ± standard deviation values for Scotchbond Universal on sound dentin reported in the literature (49.8 ± 5.3 MPa) [The sample size calculation was performed online (5.3 MPa) ,30. To d5.3 MPa) 3PO4, 20.10 mM KCl, 11.90 mM Na2CO3, and 1.98 mM CaCl2, pH of 6.7, 10 mL per specimen) for 60 min [Thirty-two prepared human molars were randomly selected to simulate erosive demineralization. Before erosive cycling, the lateral and root areas were covered with two layers of nail varnish to allow erosive demineralization only on the occlusal surface. The specimens were exposed to an erosive cyclic demineralization and remineralization procedure by immersion in a soft drink  4 times daily for 90 s each (10 mL per specimen) for 5 days ,32. The r 60 min . The rem2  [The universal adhesives were only applied in the self-etch mode and according to the manufacturer instructions . For allUT, USA) ,15 in ac2, Valo, Ultradent Product, Salt Lake City, UT, USA). A single trained operator performed all restorative procedures  applied in 2 mm increments and each increment was light-cured for 40 s , and rotated in 90° angles to obtain resin–dentin bonded sticks with a cross-sectional area of approximately 0.8 mm², measured using digital calipers  to calculate the bond strength in MPa. All resin–dentin bonded sticks that underwent pretest debonding during specimen preparation were recorded for each tooth.A total of 26–32 resin–dentin bonded sticks were obtained per tooth including the pretests debonding. The resin–dentin bonded sticks were divided as follows: for the nanoleakage test, 3 resin–dentin bonded sticks per tooth from each experimental condition group were tested after 24 h or 2 years of water storage; for microtensile bond strength, the remaining resin–dentin bonded sticks were tested after 24 h or 2 years of water storage. The distilled water was changed monthly.After 24 h or 2 years of water storage, the resin–dentin bonded sticks were attached to a modified Geraldeli device  using a The failure mode of each resin–dentin bonded stick was observed using a digital microscope  and classified as cohesive ((C), failure exclusively within the dentin or resin) or adhesive/mixed ((A/M), adhesive or mixed failure inside any of the bonded substrates). For statistical analysis, specimens with pre-test failures (PF) were included in the tooth mean with a value of 4.0 MPa .Three resin–dentin bonded sticks per tooth for each storage condition that had not been used in the microtensile bond strength test were placed in an ammoniacal silver nitrate solution in the dark for 24 h, rinsed in distilled water, and immersed in a photo-developing solution for 8 h under fluorescent light ,37. The Three images of each bonded stick were captured, including one at 0.3 mm to the right of center, one at 0.3 mm to the left of center, and one at the center. ImageJ software was used to determine the relative nanoleakage percentages along the adhesive and hybrid layers in each specimen .® , version 17.0 .The microtensile bond strength and nanoleakage data for all the resin–dentin bonded sticks from the same hemi-tooth were averaged for statistical purposes. Thus, the experimental unit in this study was the hemi-tooth. After evaluating the normality (Kolmogorov–Smirnov) and equality of variances (Bartlett), the microtensile bond strength (MPa) and nanoleakage (%) data for each adhesive were subjected to three-way repeated measures ANOVA (dentin vs. treatment vs. storage time) and Tukey’s test. The level of significance was set at 5%. All analyses were performed using SPSSp < 0.000001), and for the main factors’ dentin, treatment, and time (p < 0.000001).Approximately 80–104 resin–dentin bonded sticks per experimental group were evaluated for microtensile bond strength. The most common failure pattern in all the experimental groups was the adhesive/mixed-type failure . Few prep > 0.05).For Prime & Bond Active and Scotchbond Universal adhesives, extra HL did not significantly increase the microtensile bond strength values in the immediate group when applied to sound and eroded dentin, with the exception of Prime & Bond Active in eroded dentin (p = 0.0001). In contrast, Scotchbond Universal did not significantly decrease the microtensile bond strength after two years of water storage, compared with the immediate results for the extra HL group (p < 0.05).For Prime & Bond Active, after two years of water storage, a significant decrease in the microtensile bond strength was observed for the control and extra HL groups, compared to the immediate results in both substrates (p = 0.0001). In all cases, the microtensile bond strength values for sound dentin were higher than those for eroded dentin (p = 0.000001).After two years, the application of extra HL produced significantly higher microtensile bond strength values for both adhesives compared to the control group (p < 0.00001), and in the main factors’ dentin, treatment, and time (p < 0.0001).For nanoleakage, 24 resin–dentin bonded sticks per experimental group was evaluated. Nanoleakage data are presented in p > 0.05). In contrast, using extra HL on eroded dentin significantly decreased silver nitrate uptake values in the immediate group for both adhesives (p = 0.0001).For Prime & Bond Active and Scotchbond Universal adhesives, using extra HL did not significantly decrease silver nitrate uptake values in the immediate group when applied to sound dentin (p = 0.0001). However, when an extra HL coat was applied, a significant increase in the silver nitrate uptake values compared with the immediate group was only observed for Prime & Bond Active on eroded dentin (p = 0.0001).After two years of water storage, in both substrates and for both adhesives, a significant increase in the silver nitrate uptake values was observed in the control groups, compared with the immediate results (p = 0.0001). In all cases, the silver nitrate uptake values for sound dentin were lower than those for eroded dentin (p = 0.000001).After two years, the application of extra HL resulted in significantly lower silver nitrate uptake values than in the control group for both adhesives  . Once thAlthough there was a significant decrease in bond strength after two years in the control and extra HL groups for Prime & Bond Active, compared to the immediate results, a significant decrease was not observed for Scotchbond Universal with extra HL. One difference between Scotchbond Universal and Prime & Bond Active is the presence of a polyalkenoic acid copolymer in Scotchbond Universal. Initially, the rationale for using the polyalkenoic acid copolymer was to provide better moisture stability . HoweverIt was observed that, despite the additional hydrophobic resin layer that indicated reduced silver nitrate uptake values and improvements in bond strength, which allowed the formation of a more durable resin–eroded dentin interface, some degradation of the hybrid layer was still observed after two years of water storage. Therefore, another simple means of improving the adhesive properties of hydrophobic coatings is to incorporate bioactive materials in their contents, including different bioglass, or even more promising materials, such as phosphorene and borophene ,66,67,68It is important to mention some limitations of the present study. One of them is related to the fact that, despite the promising results observed in the present study, this is an in vitro study, which only partially simulated the intraoral conditions. Therefore, future clinical studies evaluating the effect of an additional hydrophobic resin layer associated with universal adhesives in the restoration of eroded teeth should be carried out. The second limitation is the fact that only two universal adhesives were evaluated. As universal adhesives could be considered a class of materials with several differences regarding their composition , future An additional hydrophobic resin coating increased the bond strength and reduced nanoleakage in eroded and sound dentin in the immediate time when compared to the application of universal adhesives only. However, this additional hydrophobic resin coat significant decreased the degradation of dentin after 2 years of water storage, mainly for eroded dentin, the most degradable substrate. Therefore, this strategy could be considered a feasible alternative to improve the adhesive properties of eroded dentin after two years of water storage. Further clinical studies or hydrophobic coatings containing bioactive materials are needed as alternatives to improve the adhesive properties of eroded dentin.


Given the crucial role of mitochondria as the main cellular energy provider and its contribution towards tumor growth, chemoresistance, and cancer cell plasticity, mitochondrial DNA (mtDNA) could serve as a relevant biomarker. Thus, the profiling of mtDNA mutations and copy number variations is receiving increasing attention for its possible role in the early diagnosis and monitoring therapies of human cancers. This applies particularly to highly aggressive pancreatic cancer, which is often diagnosed late and is associated with poor prognosis. As current diagnostic procedures are based on imaging, tissue histology, and protein biomarkers with rather low specificity, tumor-derived mtDNA mutations detected from whole blood represents a potential significant leap forward towards early cancer diagnosis. However, for future routine use in clinical settings it is essential that preanalytics related to the characterization of mtDNA in whole blood are thoroughly standardized, controlled, and subject to proper quality assurance, yet this is largely lacking. Therefore, in this study we carried out a comprehensive preanalytical workup comparing different mtDNA extraction methods and testing important preanalytical steps, such as the use of different blood collection tubes, different storage temperatures, length of storage time, and yields in plasma vs. whole blood. To identify analytical and preanalytical differences, all variables were tested in both healthy subjects and pancreatic carcinoma patients. Our results demonstrated a significant difference between cancer patients and healthy subjects for some preanalytical workflows, while other workflows failed to yield statistically significant differences. This underscores the importance of controlling and standardizing preanalytical procedures in the development of clinical assays based on the measurement of mtDNA. Pancreatic cancer is an extremely malignant and aggressive disease with a limited prognosis and rising incidence, being responsible for an estimated 48,220 deaths in the United States in 2021 . This unHuman mitochondria are intracellular organelles composed of eukaryotic, archaeaic, bacterial, and phagic components and play a major role in the production of cellular energy in the form of ATP through the process of oxidative phosphorylation. While the main part of our genetic information is localized in the nucleus, mitochondria contain their own genome within their matrix . MtDNA iMtDNA content and alterations have been shown to be associated with a wide range of diseases, including cancer development and progression and cardiovascular diseases that are associated with ischemic or hypoxic conditions ,35,36,37In light of ongoing studies, mtDNA used in liquid biopsy could potentially serve as a new biomarker for pancreatic cancer patients. As mentioned above, patients are mainly diagnosed in advanced disease states. Through improved diagnosis, prognosis, and therapy monitoring , the characterization of mtDNAcn or other potential alterations in mtDNA could, in the future, improve the overall management of pancreatic carcinoma patients.However, in the many years that it has taken to develop the above-mentioned FDA-approved assays, it has become clear that the development of clinically meaningful liquid biopsy assays, as well as assays that exhibit the necessary sensitivity and specificity required for implementation as a routine diagnostic test, necessitates the rigorous optimization and standardization of preanalytical procedures. It is now well understood that many factors in the long chain of preanalytical steps between sample collection and analysis can greatly impact measurements. Therefore, in order to ensure that laboratory results give the most accurate portrayal of the conditions in the vascular system of the patients ,44,45, iMuch research has been done on the preanalytical standardization of cfDNA measurements ,48,49,50Therefore, in this work we compared the yield between two mtDNA extraction methods and assessed several important preanalytical steps such as the use of two different blood collection tubes, different storage temperatures, and yields in plasma or whole blood. To identify analytical and preanalytical differences, these variables were all tested in the blood of healthy subjects and pancreatic carcinoma patients. Our study setting is a substantial precondition for a standardized and quality-controlled use of methods in clinical biomarker studies.n = 150) and ACCEPT (n = 119) studies (published therapy studies in which patients with metastasized pancreatic cancer were recruited) were used [The blood of 10 healthy individuals and 10 patients who were treated for pancreatic cancer at the Department of Internal Medicine III and Comprehensive Cancer Center at the University Hospital Munich-Grosshadern was obtained after written consent was given by the blood donors. The study was approved by the ethics committee of the Ludwig-Maximilians-Universität München (Project Nr: 21-0707). Furthermore, 10 samples of the RASH  in whole blood vs. plasma, (ii) when stored at room temperature (RT) vs. frozen at −20 °C for six days prior to processing, (iii) when collected in PAXgene Blood DNA vs. EDTA tubes, (iv) when isolated with two different extraction kits, and (v) in samples collected in 2014–2015 vs. samples collected in 2021. nDNA was measured as a control in selected experiments.® KALIUM-EDTA, Sarstedt AG & Co., Nümbrecht, Germany) of 9 mL and two PAXgene™ Blood DNA tubes  of 8.5 mL. After two hours of storage at room temperature one EDTA and one PAXgene Blood DNA tube each were spun at 1600× g for 10 min at room temperature. Plasma was removed in falcon tubes, without disturbing the buffy coat, and respun at 2800× g for 15 min to remove residual cellular debris. Whole blood and obtained plasma of both tubes was sub-aliquoted into 1.5 mL microcentrifuge tubes  and stored at −20 °C.A total of 35 mL of venous blood was collected by venipuncture in two K3-EDTA tubes . The manual kit is based on three main steps: (i) cell lysis and neutralization, (ii) plasmid DNA binding on the silica membrane, and (iii) finishing with washing steps and elution in 50 μL of elution buffer. Of the sample, 1 mL was processed for the plasmid kit in duplicate, and 0.5 mL was used for the automated kit.For all samples in this study, mtDNA was isolated with a magnetic bead-based, partially automated extraction method using the Wizard® System . The reaction mixture consisted of 1 μL sample, 800 μL master mix, 680 μL nuclease free water, and 20 μL of forward and reverse primer (20 μM) each. The primers used for mtDNA were MT-TL1: F1, 5′CACCCAAgAACAgggTTTgT-3′; R1, 5′-TggCCATgggTATgTTgTTA-3′. The primers used for nuclear DNA were β-globin: F1, 5′-gAAgAgCCAAggACAggTAC-3′; R1, 5′-CAACTTCATCCACgTTCACC-3′. These primers were synthesized by TIB Molbiol . PCR conditions were set to 95 °C for 60 s, followed by 40 cycles of 15 s denaturation at 95 °C, 30 s annealing at 58 °C, followed by 30 s extension at 60 °C. The absolute concentration of the target gene was calculated using a standard curve, which was based on a dilution series measured with qPCR. The mentioned standard curve and copy number calculation was performed as follows: PCR was performed using MT-TL1 and β-globin primers, and the DNA cleanup was performed using the DNA CleanUp Monarch DNA Kit. Fragment size distribution was assessed using the Bioanalyzer DNA 1000 kit , and the DNA enrichment was measured using the Qubit dsDNA HS Assay. The copy number was then calculated using the mtDNA concentrations and the base pairs were determined by the Bioanalyzer (Number of copies = (amount × 6.022 × 1023)/(length × 1 × 109 × 660)). Samples were measured in triplicates for both assays, enabling the calculation of the average mtDNA to nDNA ratio.nDNA and mtDNA was amplified using a 96 well-plate on the LightCycler 480www.graphpad.com (accessed on 22 June 2022)) Grubbs’ test was used to identify outliers. Only p-values < 0.05 were considered to be statistically significant.All statistics and data visualization were performed using GraphPad Prism version 9 for Windows  whole blood contains significantly higher levels of mtDNA than plasma, while ratios of mtDNA/nDNA in plasma are generally higher than in WB, which may be due to a smaller amount of nDNA in plasma or the underestimation of nDNA levels due to inefficient extraction of nDNA by kits tailored for mtDNA extraction; (ii) higher yields are generally achieved when samples were stored at −20 °C before processing; (iii) PAXgene tubes may be better suited for extractions at RT vs. EDTA tubes. Apart from this, there were no significant differences observed between EDTA and PAXgene tubes; (iv) extraction with the Maxwell Kit out of WB delivers higher yields of nDNA in general, but also higher mtDNA levels overall, which may be essential for meeting the high input demands of downstream analyses, such as NGS; (v) the QIAprep Kit yields less nuclear DNA and therefore has a higher ratio of mtDNA/nDNA but captured less absolute mtDNA than the Maxwell Kit. Choosing between these two kits depends on the aims of the experiment in question, e.g., the necessity of a high ratio of mt/nDNA and primarily “pure” mtDNA, on the impact of the presence of nDNA on further processing, or on the importance of a high mtDNA yield for downstream experiments; (vi) In view of the fact that stable sample material is indispensable for the reliability of analytical conclusions, it is also necessary to take into account that the storage time of blood between two samples can cause variations in copy number comparisons; and lastly (vii) when comparing mtDNA levels in pancreatic cancer patients vs. healthy subjects under various preanalytical conditions, more mtDNA is yielded when extracting at RT or from plasma. This indicates that there is more extracellular mtDNA in the blood of pancreatic cancer patients. However, it is also clear that this correlation only exists when specific preanalytical procedures are followed. Thus, a conclusion cannot be confidently drawn. This shows that future studies on the standardization of mtDNA extraction and all relevant preanalytical procedures are crucial in order to maximize the reliability of measurements and allow complete access to non-invasively investigate changes in the mitochondrial genome.


All women were admitted to hospital up to 72 h after birth. On admission, a qualitative fetal fibronectin test was performed in cervicovaginal secretions and transvaginal sonographic measurement of cervical length was carried out. The primary outcome measure was delivery within 5 days of presentation. RESULTS: Among the women who delivered within 5 days after admission, 65.2% had positive fFN assessment, 43.5% had cervical length below 26 mm, 52.2% had the age > 32.5 years, 34.8% were nulliparous and 56.5% had gestational age ≥ 275 days. Logistic regression analysis demonstrated that significant contributors to the prediction of delivery within 5 days were fibronectin positivity, cervical length ≤ 26 mm, maternal age > 32.5 years and gestational age ≥ 275 days, with no significant contribution from parity. (3) Conclusions: Qualitative fetal fibronectin test and transvaginal cervical length measurement in term pregnancies are useful tests for predicting spontaneous onset of labour within 5 days. It helps women and healthcare providers to determine the optimum time for hospital admission.(1) Objective: This study aimed to determine whether qualitative fetal fibronectin and transvaginal sonographic measurement of cervical length are effective in predicting delivery in term pregnancies within 5 days of the test. (2) Methods: We examined 268 women with singleton pregnancies presenting themselves at 37 The initiation and progress of labour is beyond voluntary control and under the influence of multiple physiological, psychological, and iatrogenic factors. The question concerning the time of delivery is important for the woman and her family as to the obstetrics team, placing a considerable strain on all those mentioned before . KnowingThe appraisal of impending labour is commonly done by a combination of clinical symptoms, abdominal pain and uterine contractions, and clinical assessment of cervical dilatation. However, there is no method to accurately predict the time of the onset of labour at term. With a diagnosis of pre-labour, “false” labour, or latent phase of labour, most women will be discharged after assessment, awaiting at home the spontaneous onset of labour, whereas some women will be hospitalized for monitoring under the presumption of impending onset of labour. Consequently, women removed from their communities, particularly when psychological, financial, and transport issues confront the clinical situation, may undergo prolonged hospitalization with increased administrative and economic costs, in addition to heightened maternal stress and morbidity due to extended hospital admissions ,4,5.Therefore, ongoing attempts aimed at identifying methods to determine with precision the onset of spontaneous labour and, consequently, the optimum time for hospital admission. To date, the Bishop score is the single established tool used in clinical practice to predict labour, despite concerns regarding the reliability and interobserver variability of the method ,7. Thus,To this end, the objective of this prospective study was to assess the efficacy of fFN test and ultrasound measurement of cervical length (mm) in predicting delivery within 5 days from the test, in women with term pregnancies and pre-labour from north-east Romania.This was a prospective observational study of sonographic measurement of cervical length and determination of fetal fibronectin positivity in cervicovaginal secretions in women with term pregnancies and pre-labour presenting themselves to the triage of the Obstetrics and Gynecology Hospital Cuza Voda from March 2019 to February 2022. The pre-labour or “false” labour was defined as irregular uterine contractions with or without lower abdominal pain and/or back pain not associated with progressive changes in the cervix dilatation.+0 and 40+4 weeks of gestation. Exclusion criteria included: (a) women in active labour, defined as regular, painful uterine contractions resulting in progressive cervical effacement and dilatation of 3 cm or more, (b) women with severe obstetric and medical conditions, (c) planned induction of labour or Cesarean section delivery, (d) fetal anomalies, (e) history of cervical insufficiency, (f) vaginal spotting, (g) ruptured membranes, and (h) history of previous cervical surgery ). Those women who had sexual intercourse, vaginal examination, or transvaginal ultrasound within 24 h before presentation.The inclusion criteria were: age over 18 years, singleton, cephalic, term, uncomplicated pregnancy, planned for vaginal delivery, and intact membranes. Gestational age was calculated from the menstrual history and by an ultrasound scan in early pregnancy. In the study, we included women with pregnancies between 37The eligible women were informed about the purpose of the study and signed an informed consent before participation. This study was conducted in accordance with the Declaration of Helsinki, and approved by the local institutional ethics board . Upon hospital admission, women underwent a clinical assessment that included a complete history and clinical examination with an assessment of fetal wellbeing. All women underwent hospitalization up to 72 h after birth, according to our maternity protocol.On admission to hospital, a sterile vaginal speculum examination was performed to assess the cervical changes and confirm the integrity of the membranes. For women who agreed to participate in the study, a specimen of cervicovaginal secretions was collected from the posterior fornix or endocervix, and qualitative detection of fetal fibronectin was performed as described by the manufacturer . Since the test was qualitative, with a positive cut-off value set at 50 ng/mL or greater, the results of the test were recorded as binary, positive and negative. Subsequently, a digital examination was performed by the attending clinicians, and patients with a cervical dilatation of 2 cm or more were excluded from further analysis. After the digital examination, transvaginal sonography was performed and the cervical length was measured by appropriately trained sonographers, from the internal ostium to the external ostium. Three measurements were performed for the cervical length, and the shortest one was taken into consideration for analysis. The results of the fFN tests and TVCL were recorded on an Excel sheet. Demographic and clinical information were extracted from the medical records and recorded onto the same Excel sheet as the results of the fFN tests and TVCL. The study outcome was delivery within 5 days of presentation.t test was used to compare normally distributed continuous variables and the Mann–Whitney U test was used for variables without normal distribution. In order to identify the cut-off values of continuous variables we used the ROC analysis, by calculating the AUC value and the optimal sensibility and specificity. The Chi-square test and Fisher approximation method were used to compare categorical variables. Univariate analysis was performed for each recorded variable, and variables with p-value < 0.01 in univariate analysis were included in multivariate analysis. For adjusted effect we used the logistic binary regression, Forward LR, in 4 steps. The level of statistical significance was defined as p-value < 0.05.Statistical analysis was performed using SPSS Software Version 27.0 for Windows . Continuous variables were presented as mean ± standard deviation and categorical variables as frequencies and percentages. Student’s +0 weeks gestation. All women had fFN assessment followed by transvaginal sonographic measurement of cervical length at admission. Delivery within 5 days of presentation occurred in 92 (34.3%) cases. We recruited 268 consecutive pregnant women with abdominal pain, uterine contractions, and gestational age ≥ 37We investigated the prognostic factors for delivery in 5 days after admission; the most important are the positive fFN assessment and the cervical length; in addition to these, however, there may be other elements that favor delivery within 5 days after admission, possibly cumulated with the first two.Positive fFN assessment seems to have a definitory role in identifying the spontaneous onset of labour within 5 days: it was obtained in 62.5% such cases, compared with only 4.5% cases among those with spontaneous onset of labour in more than 5 days and has a very high OR (Odds Ratio) coefficient (39.375) .Cervical length at admission was significantly shorter at women who delivered within 5 days after admission (28.83 ± 8.274) compared with the others (32.95 ± 6.371); the ROC analysis revealed the cut-off value of 26 mm, associated with an AUC coefficient of 0.628  and a goWomen who delivered within 5 days after admission were also significantly older than the others, with an average age of 31.48 ± 6247 years and a median of 33 years, compared with 29.14 ± 5491 years and a median of 29.50 years; the ROC analysis revealed the cut-off value of 32.5 years, associated with an AUC coefficient of 0.638 and also a good specificity (0.818) . Women oParity is not statistically significant correlated to delivery within 5 days after admission , even ifPatients who delivered within 5 days after admission have also had a significantly higher gestational age (273.52 ± 7.754 days) compared with the others (266.64 ± 5.987 days). The optimal cut-off value seems to be of 274.5 days, associated with an AUC coefficient of 0.754 and again a very good specificity (0.909) . 56.5% wp/(1 − p) = −6418 + 3910 * (positive fFN assessment) + 1664 * (Age > 32.5) + 1207 *  + 3301 * .Therefore, the prognostic factors for delivery within 5 days after admission identified through univariate analysis were the positive fFN assessment, cervical length at admission ≤ 26 mm, woman’s age > 32 years and gestational age at admission ≥ 275 days. Their multivariate analysis through binary logistic regression shows that all these predictors are important and their combined presence improve the prognostic chances for delivery within 5 days after admission from 65.7% to 88.1%. The probability of delivery within 5 days after admission can be calculated using the equation: Ln  There is no laboratory or clinical test available up to date to predict with certainty the time of labour onset; (ii) Women with pre-labour or “fake labour” are transferred from their communities to regional centres in the cities, sometimes hundreds of kilometers away, and remain as inpatients for several days, increasing the health care costs and with consequences on the health and economics of women and their families; (iii) These tests are commercially available and successfully used clinically for prediction of preterm birth.Several previous studies have assessed the effectiveness of fetal fibronectin testing in term pregnancies. In agreement with our findings, a study of 75 pregnant women with term pregnancies from remote areas of Australia, found that the presence of fFN in cervical secretions was associated with increased odds of delivery within 7 days. However, this group found that fFN absence did not reliably exclude the onset of birth . In accon = 17), and the analysis was only descriptive. Taken together, these results suggest that delaying women’s transfer based exclusively on the fFN findings might result in some women birthing in their home communities.In contrast with our findings, a recent study by Healy et al. found that the presence of fetal fibronectin in cervical secretions did not predict term delivery . HoweverAlthough TVCL is a reproducible and cost-effective test, studies demonstrate conflicting results regarding its predictive accuracy of term birth.Our findings suggest that the risk of a patient giving birth in the first 5 days after the measurement is 3.343 times higher if she has a TVCL below 26 mm compared to the opposite situation. Similar to our findings, a study conducted on 199 low-risk antenatal women reported that TVCL measurement between 37 to 40 weeks is a useful test for predicting delivery within 7 days . In thisSeveral observational studies have noted that the combination of fFN and TVCL assessment may improve the prediction of spontaneous preterm birth in women with preterm symptoms. In a prospective study of 665 women with threatened preterm labour, van Baaren et al. found that women with a TVCL ≥ 30 mm or those with a CL 15–30 mm and a negative FFN result were at low risk (defined as 5%) of spontaneous delivery within 7 days . On the To our knowledge, this is the first study to interrogate the clinical applicability of combined fFN test and TVCL in clinical decisions regarding hospitalization or transfer of pregnant women at term for labour and delivery. Multiple logistic regression analysis demonstrated that the prediction accuracy for birth within 5 days of hospitalization is 88.1% in the presence of all 4 identified prognostic factors, i.e., positive fFN assessment, cervical length at admission ≤ 26 mm, woman’s age > 32 years and gestational age at admission ≥ 275 days.In this study, we found that a risk prediction model including qualitative fetal fibronectin test, transvaginal sonographic cervical length measurement and clinical risk factors showed promising performance in the prediction of spontaneous term delivery within 5 days of the test. Testing fFN in cervicovaginal secretion at term is indicative of spontaneous labour, but its absence does not fully exclude the possibility of the onset of labour.Further evaluation of the risk prediction model in clinical practice is required to determine whether the risk prediction model improves clinical outcomes if used in practice. Validation of this procedure in accurate prediction of spontaneous onset of labour will not only reduce the duration of hospitalization and lower the expenses in maternities but might also help to avoid the adverse effects of prolonged antepartum hospitalization on the postpartum quality of life.


Difficult laparoscopic cholecystectomy (LC) due to acute cholecystitis (AC) increases the risk of bile duct injuries and postoperative complications. Here, we added the lateral dorsal infundibular approach as an initial surgical maneuver during LC to improve outcomes.We describe the detailed technical procedure of the lateral dorsal infundibular approach in patients with AC resulting in difficult LC. This technique was developed after nearly 10 years of experience in laparoscopic surgery, and has been routinely used in the past 5 years. We also retrospectively analyzed the perioperative data for 469 patients with difficult LC.A total of 469 patients with AC received difficult LC between July 2016 and June 2021, of which 438 (93.4%) performed a lateral dorsal infundibular approach. Sixty-four patients (13.6%) had variations of the hepatic bile duct and cystic duct according to preoperative magnetic resonance cholangiopancreatography, 438 patients (93.4%) received elective surgery, 31 (6.6%) received emergency surgery, and 10 (2.1%) underwent conversion. There was no postoperative bile leaks and no bile duct injuries in the described technique.During difficult LC, the critical view of safety can be gradually achieved by changing the surgical approach to achieve cholecystectomy. Changes in the sociodemographic structure have led to a gradual increase in the incidence of acute cholecystitis (AC), due to the occurrence of gallstone disease among the aging population. Previous studies have shown that increasing age is significantly related to increases in surgical complications and perioperative mortality . SurgeonLaparoscopic cholecystectomy (LC) has been widely recognized as a safe and effective treatment for gallstones. Its faster recovery, less pain, and shorter hospital stay compared with open surgery mean that LC has been the standard surgical treatment . The iniAC is caused by excessive gallbladder (GB) enlargement, edema, and congestion, making the Calot’s triangle relationship unclear. It is difficult to establish a “critical view of safety” (CVS) during LC, and the chances of bile duct injuries (BDI) and bleeding are increased, making it difficult to complete LC successfully. Conversion to laparotomy is then usually recommended as an important and indispensable bail-out procedure for difficult LC . HoweverHere, we report the technical details of the lateral dorsal infundibular approach, developed over the past 10 years, which has demonstrated success during difficult LC for the treatment of severe inflammation or gangrenous cholecystitis. The main aim of the procedure is to avoid BDI by using laparoscopic STC technique to explore and mark the infundibulum to determine the initial anatomical landmarks, from the lateral submucosal approach to the dorsal, until complete circumferential dissection of the infundibulum allows CVS, and the GB can then be removed in sections. We evaluated this technique in 469 cases of difficult LC.This study was approved by the Ethics Committee of the 925th Hospital of the Chinese People’s Liberation Army. In this single-center study, we retrospectively analyzed the medical records of 469 patients with pathologically confirmed AC who underwent difficult LC between July 2016 and June 2021. These patient records were reviewed by two senior surgeons, and only patients who met the Tokyo Guidelines 2018 criteria for the diagnosis of AC and performed the lateral dorsal infundibular approach were included in the analysis. Patients with gallbladder cancer confirmed by postoperative pathology were excluded. The data collected included the patients’ demographic characteristics , and preoperative ultrasound and magnetic resonance cholangiopancreatography (MRCP) results. Perioperative data included the timing of surgery (elective/emergency), conversion, infundibulum management (fenestrating or reconstituting), bile leaks, retained stones, operation time, blood loss, hospitalization duration, and follow-up. The follow-up period ended in November 2021. Statistical analysis of the data was performed by SPSS 25.0 software, all data was analyzed by descriptive statistics.The camera port position is located close to the bottom of the navel, in line with operating habits and taking account of the aesthetic results. According to the difficulty of operation, three or four ports were selected. The main operation port is a 10-mm trocar placed on the right side of the round hepatic ligament under the xiphoid process, and the other auxiliary ports are one or two 5-mm arc-shaped trocars placed under the ribs around the operating area  (including endoscopic sphincterotomy and temporary stent placement) according to the postoperative drainage volume. Following these treatments, bile leaks usually resolve within 1–2 weeks.We retrospectively analyzed 469 cases of difficult LC. The patients included 176 men (37.5%) and 293 women (62.5%), aged 34–87 years (mean 55.1 years). The postoperative pathology showed AC in all cases, including 215 cases (45.8%) of gangrenous cholecystitis. All patients underwent preoperative ultrasound and MRCP. The thickness of the GB wall measured by ultrasound was 4–10 mm in 361 patients (77.0%) and > 10 mm in 108 patients (23.0%). Sixty-four patients (13.6%) had variations of the hepatic bile duct and cystic duct found by MRCP. Overall, 438 patients (93.4%) received elective surgery, 31 (6.6%) received emergency surgery, and 10 (2.1%) required conversion. The described technique was completed successfully in 434 patients (92.5%), 13 patients (2.8%) underwent fenestrating STC, and 22 (4.7%) underwent reconstituting STC. Postoperative bile leaks occurred in four patients (0.9%) , of which three cases healed within 1–2 weeks after ERCP. The remaining patient who underwent reconstituting STC had bile leaks but refused ERCP treatment, and finally healed spontaneously after continuous drainage for 2 months. There were no retained stones after surgery. The mean operation time was 117.0 ± 34.5 min, the mean blood loss was 146.9 ± 53.2 ml, and the mean hospitalization duration was 7.6 ± 2.6 days. There were no injuries to blood vessels or bile ducts, no reoperation, no readmissions, and no deaths.The rapid development of laparoscopic surgery has led to increasing reports of difficult LC worldwide. Consensus guidelines from the 2018 Boston Conference and the Tokyo Guidelines recommended standardized safety procedures, emphasizing the exposure of Rouviere’s sulcus and efforts to create CVS. If the CVS cannot be achieved due to scar formation or severe fibrosis during the operation, or if the Calot’s triangle is shrinking and the boundary is unclear, a bail-out approach should be considered, including conversion, STC, or the fundus-first technique. The common point of both STC and the fundus-first technique is the retention of the infundibulum. The advantages of the technique include no dissection of Calot’s triangle, reduced bleeding, shorter operation time, and avoidance of BDI. However, the disadvantages of both the fenestrating and reconstituting techniques include postoperative bile leaks . Even loThe initial method of the infundibulum side boundary is used to reduce the dissection of Calot’s triangle and avoid the occurrence of BDI . Our tecBDI is the most serious complication of LC and presents a problem for hepatobiliary surgeons throughout their learning curve. The reported incidence of BDI was 0.23%, and although the incidence has declined in recent years, it remains higher following laparoscopic compared with open cholecystectomy . BDI assPreoperative examination is important to realize the above steps, including safe resection of the infundibulum, no BDI, and a low conversion rate. Detailed imaging examinations are critical for surgical planning, especially in cases of difficult GB assessments, such as patients with Mirizzi’s syndrome, aberrant hepatic ducts/cystic duct insertions, and rare cancers. Preoperative ultrasound can provide an initial impression, including diagnosis of gallstones, measurement of swelling, and GB wall thickness. However, we consider MRCP to be the most important and effective method for assessing the severity of cholecystitis inflammation and the extrahepatic bile ducts . MagnetiNone of the patients in the current study had developed BDI by 5 years after difficult LC using the described technique. This method thus has several advantages (1) Surgical preservation of the cystic plate mucosa is simple and quick, saving operating time for subsequent procedures, and also reduces bleeding because the plate/sheath system of the liver is not penetrated. (2) Exploration of the infundibulum cavity not only avoids the occurrence of residual stones, but also helps to confirm the overall shape from the inside out, to lay the foundation for the next step of dissection. (3) The method can effectively avoid damage to blood vessels and hepatic bile ducts, and thus avoid BDI.Minimally invasive surgery has great benefits and is extremely attractive to patients and surgeons; however AC presents a challenge for surgeons carrying out LC. We believe that after completing the laparoscopic learning curve, surgeons can innovate and explore according to the patient’s actual condition, notably in the context of the rapid development of laparoscopic technologies and the integration and clinical application of various advanced technologies such as digital medicine and artificial intelligence. During difficult LC, CVS can be gradually achieved by changing the surgical approach and safely peeling off the infundibulum to achieve cholecystectomy.


Endothelial progenitor cells (EPCs) and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase enzyme activity may affect the vessel wall andhave a role in development of aortic aneurysms. EPCs originate fromhematopoietic stem cells and can be enumerated from peripheral blood samplesby flow cytometry. In this study, we aimed to evaluate the relation of EPCnumber and NADPH oxidase enzyme activity in the development of thoracicaortic aneurysm (TAA).Patients with TAA (n=30) and healthy individuals without TAA were included in our study. Characterization and enumeration of EPC fromperipheral blood samples were performed by flow cytometry with panelsincluding markers of EPCs (CD34/CD133/CD309/CD146/CD144). Additionally,NADPH oxidase enzyme activity (capacity) was also measured by thedihydrorhodamine 123 (DHR 123) test.P<0.01).Additionally, patients with TAA presented significantly lower NADPH oxidaseactivity by DHR assay than healthy controls (P<0.01).The enumeration of EPC with CD34+/CD146+ marker showed that the number ofmean EPC/106 cells was increased in the patient group (41.5/106 cells), butnot in the control group (20.50/105 cells) (Our results showed that the number of EPCs is significantly higher in aorticaneurysm patients and may have a role in disease progression. The crosstalkbetween NADPH oxidase enzyme capacity and EPC number may be useful as aparameter to explain the clinical progression of TAA. TAAs areclinically classified into three groups as syndromic , familial (non-syndromic), and sporadic.Thoracic aortic aneurysm (TAA) is one of the major causes of morbidity and mortalityworldwide. The etiology of TAAs is probably multifactorial, and smoking, chronicobstructive pulmonary disease, hypertension, atherosclerosis, male gender, age, bodymass index, and family history are classic risk factors for this disease. Inaddition, genetic factors play an important role in the development of thedisease. It is thoughtthat the damage may happen through two main mechanisms. The two main sources ofvascular reactive oxygen products (ROS) are the nicotinamide adenine dinucleotidephosphate (NADPH) oxidase found in the mitochondrial respiratory chain and smoothmuscle cells. Superoxide, produced by NADPH oxidase enzyme, has a very shorthalf-life, is converted to hydrogen peroxide (H2O2), then tohypochlorous acid by the enzyme myeloperoxidase, and takes part inapoptosis. Thisapoptosis mediates endothelial damage. ROS production capacity of peripheralneutrophils can easily be measured by dihydrorhodamine 123 (DHR 123) assay, which ispractically used for the diagnosis of chronic granulomatous disease. NADPH oxidase enzyme has sevenisoforms in mammals — in this case, NADPH oxidase isoform (NOX) 2, expressed inendothelial cells, plays a role in the regulation of various functions of theendothelial cell and angiogenesis. It has been shown that the change in the ratio ofNOX1/NOX2 plays a role in the development of endothelial dysfunction, hypertension,and inflammation. StimulatedNADPH activity increases the amount of intracellular superoxide radical (O-.) andH2O2 in smooth muscle cells in the vessel. As a result,the ROS amount in the vessel increases, so this stimulates various intracellularsignal transduction pathways and triggers the development of TAA.Another risk factor is thought to be oxidative stress and its induced endothelialdysfunction. Oxidative stress also is known to play a role in the development ofaneurysm. The aortic aneurysm may result from the disruption between thedamage to the vessel wall and the repair mechanisms. EPCs are the main reservoircells of the endothelium, playing an important role in ensuring endothelialintegrity. Therefore, it can be thought that EPCs have a place in the pathogenesisof aortic aneurysms with impaired endothelial structure. There are few findings ofthe development of TAA disease. Although there is strong evidence that both NADPHoxidase activity and EPC balance play a role in the development of this disease,molecular mechanisms have not yet been elucidated.Endothelial progenitor cells (EPCs) have proliferation, differentiation, and tissueregeneration capacity and are precursors of endothelial cells. In the event ofendothelial damage or ischemia, secreted chemokine mediators induce EPCsproliferation from the bone marrow and migration to the inflammation site and joinsthe structure of newly formed vesselsIn the current report, we present a single-center study with 30 patients with TAA forthe enumeration of EPCs and results of DHR assay. We compared patients and controlsamples for the number of EPCs and NADPH oxidase activity since these parametershave relation with the clinical progression of TAA patients.The study consisted of 30 patients who were admitted to our clinic between 2014and 2018 and were diagnosed with TAA at the cardiology department. The localethics committee approval and patient informative consent were obtained. One hundred and fifty patients wereinvolved, but only 30 patients with aortic aneurysm who met the criteria wereincluded in this study, and 10 persons without aortic aneurysm were selected forvolunteer control group. Blood samples of the patients and the control groupwere taken for EPC and NADPH oxidase enzyme activity analyses. In this study,the tissue samples were not taken because the patients were followed up. A totalof 150 patients were evaluated with inpatient screening; 65 of these patientswere excluded because of poor image quality and because there was a differencebetween the measurements made by two cardiologists. Collagen tissue disease,malignancy, cirrhosis, hemodialysis, cardiac-S development, pulmonaryhypertension, and congenital cardiac defect were detected in 35 patients. Theremaining patients were excluded because they refused to participate in thestudy. A control group that was completely healthy and whose echocardiographicparameters were completely normal was included. Malignancy, cirrhosis,hemodialysis, cardiac-S development, pulmonary hypertension, and congenitalcardiac defect were the exclusion criteria. Electrocardiography and bloodpressure measurements were performed on each patient.Echocardiography was also performed on each patient to evaluate cardiac functionand to measure the aortic diameter. Aorta, ascending aorta, left atrium, leftventricle, and partially right ventricle were evaluated in the parasternallong-axis window. The aortic root was measured at the widest segment of themiddle sinus of Valsalva. Internal diameter measurements were taken from thewidest part of the ascending aorta. Measurements in echocardiography were takenin diastole. The aortic valve and its structure , left ventricular diameter, and mitral valve andits structure were evaluated. Aortic valve structure, aneurysm, and ascendingaortic aneurysm were used to compress other anatomic structures in the apicalfive-space window. The aortic valve was evaluated with color Doppler forinsufficiency and stenosis.Images were obtained from all patients and control groups using the VIVID S6echocardiography device by monitoring at the left lateral position.Echocardiography result was reported using routine echocardiographic windows andtechniques, and the distance of the widest part of the ascending aorta to thesinus of Valsalva was noted.. 5 µL CD34 ECD, 5 µL CD146 PC5, 5 µLCD144 PE, and 5 µL CD309 PC7 were added to 12×75 mm flow tubes.100 alkanes were added to each of the added markers and left to incubate in thedark for 20 minutes by vortexing. At the end of the period, 500 µL lysingsolution was added to each tube, and the tubes were vortexed and left toincubate in the dark for 10 minutes. At the end of the period, they werecentrifuged at 1500 rpm for five minutes at room temperature. The supernatantwas removed. Then, 2 mL of cell washing solution was added to each tube and theprevious centrifugation was repeated. After isolation, specific markers wereadded to flow tubes, vortexed, and incubated, then centrifuged, and all theprocesses performed according to the previous practice[9-. The analysis was completed by theenumeration of EPC in 1×106 cells by special gate on the flowcytometer.EPC expression was tested with CD34, CD45, CD146, CD144, and CD309 specificmarker antibodies by Navios EX flow cytometry . Total leukocytes were isolated from 100to 200 µL of blood from patients and controls using lysis of theerythrocytes in the pellet fraction with a non-fixing lysis solution, asdescribed by Köker et al.. The capacity of neutrophils to producereactive oxygen species has been tested with the DHR experiment. In this test, the isolatedneutrophils were incubated with DHR 123 , stimulated with phorbol 12-myristate 13-acetate(PMA) , and analyzed byflow cytometry with Kaluza software (Beckman Coulter). The results are shown asthe stimulation index (SI),which gives the fluorescent intensity ratio from stimulated and unstimulatedneutrophils.Total leukocytes from 100 to 200 µL of blood from patients and controlswere isolated by lysis of erythrocytes in the pellet fraction with an unstablelysis solution, as described by Köker et al.t-test. Pearson χ2analysis and Fisher’s exact χ2 test were used for comparisonof categorical data. All tests were two‐tailed, and P<0.05was considered statistically significant. The relationship between quantitativedata was evaluated by Spearman’s correlation analysis.IBM Corp. Released 2012, IBM SPSS Statistics for Windows, version 21.0, Armonk,NY: IBM Corp. was used for statistical analysis. Suitability of the data fornormal distribution was evaluated by histogram and Q-Q graphs and theShapiro-Wilk test. Continuous variables between the two groups were comparedusing the independent TAA patients , consisting of six women (meanage 58.5±12.9 years) and 24 men (mean age 58.6±8.7 years), and healthycontrols , consisting of two women (mean age58.5±19 years) and eight men (mean age 48.6±12.7 years), were includedin this study. Patient and control groups were similar in terms of age and genderdistribution.While the tricuspid aortic valve was found in 26 (86.7%) of 30 patients, the bicuspidvalve was seen in two (6.7%), and replaced aortic valve for aortic valve disease wasalso seen in two (6.7%). All the control patients had a tricuspid aortic valve. Thedemographic and clinical characteristics of the patient and control groups are shownin P<0.01). Besides, a significantdifference was found between the two groups in monocyte, hemoglobin A1C, and proB-type natriuretic peptide values. Laboratory findings of the patient and controlgroups were summarized in P<0.01 and P=0.01, respectively). In themedical treatment they received, no significant difference was detected between thetwo groups, except angiotensin receptor blocker. When the complete blood count wasevaluated between the two groups, all parameters except the monocyte ratio werefound to be similar  (reactive protein (CRP) was measured as 6.9±11.1 in the patient group and1.3±1.4 in the control group. A statistically significant difference wasfound between the groups (P=0.012) .6) cell counts were countedby flow cytometry both in control and patient groups . There wasno statistically significant difference between the number of cells expressingbetween the patient and control groups.The mean EPC numbers were found to be 41.5/10When the relationship between EPC numbers and clinical characteristics of thepatients was evaluated, no statistically significant correlation was determined.However, there was a statistically positive correlation between aortic diametermeasured by echocardiography and numbers of CD146+/CD144+ EPC. Comparing thenumber of EPC in patients with aneurysm diameter ≥ 48 mm with those withaneurysm diameter < 48 mm, CCD34+/CD146+/CD144+ cells were found to besignificantly higher in patients with aneurysm diameter > 48 mm. For theother parameters, no significant difference was found between the two groups. Flow cyNADPH oxidase enzyme capacity evaluated with SI in patients was 60.4±7.8,and in the control group it was 75.1±5.2, by DHR assay . It was [10,13,. Inflammation of the vascular layers anddestruction of extracellular matrix proteins are known to play a role in thepathogenesis of aortic aneurysm as well as hemodynamic factors[14,. ROS are involved in the pathogenesis of manychronic diseases such as atherosclerosis and hypertension. ROS are molecules that can also occur duringnormal metabolism and cause a local inflammatory response, causing progressive celland tissue death (oxidative stress). ROS level in the organism is regulated byadjusting the expression of antioxidant and oxidant enzymes to maintain homeostaticbalance. The mechanisms of ROS production in non-phagocytic cells are puzzling, butNADPH oxidase enzyme isomers play a key role. Inspired by this situation, DHR 123test was performed in phagocytic cells in our study, and when the SI values, whichare an indirect indicator of general ROS generation capacity, in both patient andcontrol groups were compared, it was found that SI values were significantly lowerin the patient group (P<0.01).The studies showed that inflammation and tissue degeneration, which are associatedwith many chronic diseases, have a great deal on the formation and progression ofaortic aneurysms[5,. In our study, the mean SI value was found to be60.40±7.86 in the patient group and 75.10±5.21 in the control group(P<0.01). This difference does not mean that ROS productionis less in the patient group. The DHR 123 test provides information about ROSproduction capacity of the NADPH oxidase enzyme in an individual. These decreasedcapacities, which also occur in aortic aneurysm patients, may need to increase theduration of the activity of NADPH oxidase enzymes in phagocytic cells and itsisomers in non-phagocytic cells, especially NOXs in vascular endothelium. Prolongedactivity of NADPH oxidase enzyme may cause tissue damage and inflammation.DHR 123 test is the most sensitive flow cytometric indicator used to detect oxidativeburst in peripheral neutrophils. This test provides information on the ROSproduction capacity of NADPH oxidase enzyme. SI values used for interpretation ofthe DHR 123 test should be between 50-100 in healthy individuals[17,. Again, smoking, hyperlipidemia,and the presence of concomitant CAD have been shown to stimulate the production ofsuperoxide in the aortic aneurysm. In our study, no significant difference was found between thepatients with TAA and with/without hypertension.There are studies in the literature evaluating the relationship between oxidativestress and hypertension, smoking, male gender, dyslipidemia, and coronary arterydisease (CAD), which are among the major risk factors for the development of aorticaneurysm[20,. Our research is the first studyin the literature to evaluate CD34+/CD146 + cells in TAA patients in addition to thefindings of other studies. Studies conducted have shown that disease progression canbe monitored by flow cytometric methods and that it can show how EPCs can play arole in the underlying pathogenesis. Experimental studies have shown that circulating endothelialprogenitor cells (or CEPC) contribute to the ongoing endothelial repair mechanism inthe affected arterial region after balloon damage and replace new dysfunctionalendothelial cells. In ourstudy, no statistically significant difference was observed in CD34+/CD309+ cellsbetween patient and control groups (P=0.27).There are several studies in the literature examining the relationship between aorticaneurysm and EPCs. In these studies, they generally investigated CD34+/CD133+ cellsin abdominal aortic aneurysm (AAA) and CD34+/CD309 + cells in ascending aorticaneurysm have shownthat CD146 is localized in the junction of two endothelial cells. CD146 regulatesthe endothelial single cell layer, controls the paracellular permeability, andprovides the connection with other neighboring cells as it is associated with actinforming the cell skeleton. There are no publications in the literature that studyCD146+ and CD144+ microparticles or cells in TAA. However, when we look at thepathophysiology of aortic aneurysm, inflammation plays an important role, andendothelial cell dysfunction occurs. CD146 level was also studied in patients with asmall number of chronic heart disease, and plasma CD146 level was found to be high.These cells are increasing in number to repair the area in case of any damage.CD34+/CD146+ cell count was found to be significantly higher in patients with TAA inour study (P<0.01). Therefore, our results are consistent withthe literature. In the study conducted by Hosseinzadeh et al., it was reported that CD144+endothelial microparticles were found to be higher in patients with vascular riskfactors  than inthose with normal cognitive function, without vascular risk factors. CD144+ cellsare involved in the adhesion of homophilic cells and again in case of any damage tothe endothelium, the number of these cells is increased to repair the damagedregion. In our study, the number of CD34+/CD146+/CD144+ cells were significantlyhigher in patients with aneurysm diameter ≥ 48 mm than in those with diameter< 48 mm (P<0.05). This may be due to the increase in thenumber of CD146+/CD144+ cells involved in regeneration in the endothelium due to theincreased diameter of the damaged region, and thus the increased number of precursorCD34+/CD146+/CD144+ cells in the blood.CD146 and CD144 are some of the components of the endothelial intercellular bindingsite. Bardin et al.In our study, monocytic blood cells were found to be significantly higher in thepatient group. While monocytic cells were significantly higher in the whole bloodcount in the patient group and CD309+ cells were higher in flow cytometric analysis,it was thought that monocytic/dendritic cells could play a role in the pathogenesisof TAA. Since monocytic cells were involved in both degenerative and regenerativeprocesses in our patient group, they were found to be significantly higher in ourpatients.. In our study, CRP level wasfound to be significantly higher in our patient group than in the control group(P<0.01).One of the risk factors of aortic aneurysm development is the high CRP level. Studieshave shown that CRP levels in patients with symptomatic and ruptured AAA aresignificantly higher than in those with asymptomatic aneurysmsOur study has some limitations. First, although the control group was chosen withsimilar characteristics to our patient group, it was lower in number than thepatient group. And secondly, this is a single-center study.Our results showed that the number of EPCs is significantly higher in aortic aneurysmpatients and may have a role in disease progression. The crosstalk between NADPHoxidase activity and EPC number may be useful as a parameter to explain the clinicalprogression of TAA. Our research is the first study in the literature to evaluateCD34+/CD146+ cells in TAA patients The presence of CD34+/CD146+ cells, which haveimportant functions in the binding region between endothelial cells, and the closeassociation of these cells with infiltration, as demonstrated in other studies,showed that CD34+/CD146+ cells may be an important marker in this disease. Our studyis a guiding light for future studies with larger numbers of patients. However,there are many unknown aspects of the pathogenesis of TAA. To diagnose this diseasewith serious complications early and to prevent its progression, these pathwaysshould be clarified. These decreased capacities of NADPH oxidase enzymes activity inTAA may be compensated by increasing the duration of the activity of it inphagocytic cells and its isomers in non-phagocytic cells, especially NOXs invascular endothelium. So, prolonged activity of NADPH oxidase enzyme with continuousROS secretions may cause tissue damage and inflammation.ww


IntroductionCoronavirus disease 2019 (COVID-19) has affected the healthcare system and the practice of physiotherapists. Telerehabilitation is an alternative method of delivering physiotherapy services. The aim of this study was to investigate physiotherapists’ knowledge, beliefs, and willingness to use telerehabilitation in Greece during the COVID-19 pandemic.Materials and methodsIn this cross-sectional study, Greek physiotherapists completed an online survey between January and February 2022. A questionnaire was distributed via the Panhellenic Physiotherapy Association (PSF). The questionnaire involved 26 items on demographic background, use of technology, overall perceptions, the experience of telerehabilitation, and their opinion on the future of telerehabilitation. The study protocol was approved by the Ethical Committee of the University of Patras, Greece.ResultsParticipants in this study were 213 physiotherapists . Most physiotherapists  were working in a private clinic in the areas of outpatient orthopedics, geriatrics, and neurorehabilitation. Overall, most participants (55%) reported increased use of telerehabilitation strategies during the COVID-19 pandemic. A total of 130 physiotherapists (n=61.3%) believed that telerehabilitation may be beneficial as a supplementary way of patient management. Greek physiotherapists made use of low-cost and easily accessible digital technologies, such as mobile phones and online meeting tools . Although most physiotherapists (79.8%) reported that they wanted to receive more information about digital technology and telerehabilitation, only 42.1% of them did intend to work remotely after the pandemic.ConclusionMost of the participants were willing to deliver physiotherapy via telerehabilitation. Specific education and training programs need to be provided to physiotherapists during and after the pandemic. Healthcare managers should consider the use of telerehabilitation and design guidelines and policies to manage telerehabilitation practices in Greece. Telehealth is the use of telecommunications and digital and virtual technology to deliver healthcare and patient rehabilitation . TelerehThe material contained within this manuscript was orally presented at the World Congress of Osteoporosis, Osteoarthritis, and Musculoskeletal Diseases on 24-26 March 2022 in Barcelona, Spain.A nationwide cross‐sectional anonymous online survey was conducted among Greek PTs between January and February 2022. An original self‐administered questionnaire was developed by the study authors (Appendices). All questions were based on the review of the literature. Pretests were performed with four individuals for linguistic purposes and to establish face validity. The questionnaire involved 25 items divided into four parts: 1) demographic data, 2) technology usage, 3) overall perceptions/experience and challenges of telerehabilitation, and 4) willingness to use telerehabilitation. PTs eligible to participate in this study were 1) licensed by the Panhellenic Physiotherapy Association (PSF), 2) actively practicing physiotherapy in Greece, and 3) voluntary informed consent to participate in the study. This questionnaire was distributed via the PSF. Participation in this study was voluntary.EthicsThe present study protocol was approved by the Ethical Committee of the University of Patras, Greece .Data analysisDescriptive statistics were used to analyze the results. Frequencies and percentages were calculated for the participants’ demographic data. Statistical results were considered significant at the 5% critical level (p<0.05). Descriptive statistics and analysis of data were performed using IMB SPPS Statistics 28.0 .A total of 213 PTs participated in the study .DemographicsThe participants had a mean age of 39.8 (SD=8) years and the majority of them were female (57.7%) Table . Most PTTechnology usageFor personal purposes, almost all participants reported that they used digital tools daily , or three to five days per week . For personal reasons, most PTs use laptops/computers , and/or phones , and live streaming platforms . For professional purposes, the majority of the participants stated that they use digital tools daily , three to five days per week 24.4.% (n = 2) used digital tools daily, 25.8% and only one never or less than once per week. They used laptops/computers , and/or mobile/phone devices , live streaming platforms such as Zoom, Skype, etc. .Barriers to telerehabilitation practiceGreek PTs record the following as significant barriers: patient confidentiality, lack of knowledge, availability of equipment, and inadequate training.Overall perceptions/experience and challenges of telerehabilitation: Overall, most participants  reported the increased use of telerehabilitation strategies during the COVID-19 pandemic. Out of 116 PTs, 52.9% started to practice some form of telerehabilitation during the COVID-19 pandemic  believed that telerehabilitation may be beneficial as a supplementary way of patient management. Greek PTs made use of low-cost and easily accessible digital technologies, such as mobile phones and online meeting tools . Although most of them  reported that they want to receive more information about digital technology and telerehabilitation, only 42.1% (n=48) of them did intend to work remotely after the pandemic.The present study showed that most of the Greek PTs practiced some form of telerehabilitation during the COVID‐19 pandemic. The majority of Greek participants expressed a positive perception of this form of rehabilitation. This finding is similar to PTs working in Kuwait . TelerehTelerehabilitation includes services such as assessment, diagnosis, prognosis, treatment, and follow-ups . The majImportant issues are data security and patient confidentiality. In the present study, most PTs who used some method of telerehabilitation did not any action regarding the security of personal data. PTs should be concerned about data security, which seems to remain a disadvantage of digitalization -24. PhysThe present study recorded that the majority of the Greek PTs were willing to use telerehabilitation in the future. This finding is consistent with previous studies ,8,10,11.Regarding the effectiveness of telerehabilitation services, the results of a systematic review showed that telerehabilitation offers beneficial clinical results regarding physical function, especially in patients with neurological conditions. Future studies are required in order to investigate the effectiveness of telerehabilitation services , as teleThis study was the first to be conducted with Greek PTs regarding telerehabilitation practices. A strength of the survey was that it was supported by the PSF, which highlights the significance of the topic. However, the sample was small compared to the number of active licensed PTs in Greece, making it not representative . Data reThis study highlights the importance of understanding PTs' perceptions of telerehabilitation in clinical practice. The present study identifies the willingness and the need for further education and training regarding telerehabilitation. The majority of Greek participants expressed a positive perception of telerehabilitation. In addition, Greek PTs want to receive more information about digital technology and continuous educational training regarding telerehabilitation practice. Researchers recommend communication between health policy, legislators, and health professionals in order to develop guidelines and manage telerehabilitation practices in Greece. Future research is needed regarding the lack of data security and knowledge on legal frameworks for telerehabilitation practices.