With LDx/NS2400 platform, TSH values were in the mean 2.09 mU/L higher; however, the difference of both results from the mean was within ±2 SD, up to 30 mU/L, and only for values above 50 mU/L did the difference become bigger. However, this has no influence on the clinical interpretation. No false negative results were observed with either of the two platforms. TSH results obtained with the LDx/NS2400 were slightly higher than those obtained with the PE/GSP; however, the recall rate was lower: 0.059% compared to 0.063%. This can be explained by the much narrower distribution of TSH values. In conclusion, both platforms are equally suitable for medium and large NBS laboratories. However, due to the more open structure the LDx/NS2400 platform has a lot of advantages compared to the totally closed PE/GSP platform.Newborn screening (NBS) for congenital hypothyroidism (CH) started in the 1970s, with the introduction of radioimmuno assays (RIA) for the measurement of thyroxine (T4), and thyroid stimulating hormone (TSH). With the development of sensitive enzyme immune assays , RIAs were replaced in the newborn screening laboratories. With the increasing number of analytes and centralization of NBS, there is a growing demand of total automation. In the course of method validation, two fully automated platforms for the determination of TSH in dried blood samples (DBS) were compared. The GSP from PerkinElmer (PE), and the NS2400 from Labsystems (LDx), together with the recommended test kits from both manufacturers. Both systems showed good performance, with recoveries, of 103.0% (LDx) and 98.5% (PE), and CVs for intra and interassay variations at various concentrations, between 4.3 and 15.7. Both assays had a good correlation (r Besides the test kits for TSH, for both platforms there are also test kits available for total tyroxine (T4), 17-hydroxyprogesterone (17-OHP), immunoreactive trypsinogene (IRT), biotinidase, galactose-1-phosphate uridyl transferase (GALT), total galactose, glucose-6-phosphate dehydrogenase (G6PD), and phenylalanine. Both systems can handle up to 25 microtiter plates at a time, with the option of continuous loading. The processing time for the different assays is also comparable, since it is mainly determined by the incubation times. Liquid handling is slightly faster on the LDx/NS2400, because it uses 8-channel pipetting technology without additional plastic pipetting tips, while the PE/GSP just uses two pipettes, one for low volumes, and one for high volumes. The major difference between the two systems is the open architecture of the LDx/NS2400. It allows to use all included components  also as stand-alone instruments, and, in addition, it also allows the use of organic solvents on the instrument. Neither are possible with the PE/GSP. In addition, the LDx/NS2400 is able to transfer liquid from one microtiter plate to another, which increases the flexibility, and prevents floating disk problems with the enzymatic assays [Newborn screening (NBS) for congenital hypothyroidism (CH) became feasible after the development of radioimmunoassays (RIA) for the measurement of thyroxine T4)  and thyr  and thysurement ,10, or esurement . Therefosurement . This stc assays .Capillary blood is taken by heel prick between 72 and 96 h of life, and dried on special blood collection devices  the so called dried blood spot (DBS) samples. TSH was measured from 3.2 mm punches from the DBS with two different test kits, in parallel, by using the GSP Neonatal hTSH kit on the GSP instrument from PerkinElmer, Turku, Finland, and with the Neonatal HTSH FEIA PLUS test kit on the NS2400 from Labsystems Diagnostics Oy, Vantaa, Finland. Both test kits were used according to the manufactures instructions.In total, 5029 normal samples from the routine newborn screening were compared. In addition, 472 samples with initial TSH values between 2 and 460 mU/L on either platform, were repeated in duplicate, on the other platform, respectively, or on both platforms. From these 472 samples, 66 were from newborns with confirmed CH. Furthermore, in total, 36 samples from external quality assessment program (EQA) were measured on the LDx/NS2400 platform. In total, 21 samples came from the german RfB , 11 from UK-NEQUAS  and 4 from CDC . The 11 samples from UK-NEQUAS were also measured on the PE/GSP platform. The other EQA samples had, unfortunately, not enough material for parallel measurements.2 = 0.8814 (2 of 0.849 with a 95% CI of 0.609–0.946 (p < 0.001), was considered as “significant relationship” [For this comparison study 5029 unselected normal samples, 430 samples with TSH > 5 mU/L and 66 samples of newborns with confirmed CH were used. All samples were measured on both platforms with the Labsystems test kits on the NS2400 (LDx/NS2400), and the PerkinElmer test kits on the GSP (PE/GSP). The two assays showed a good correlation with r= 0.8814 . ComparaThe TSH values were slightly higher with LDx/NS2400, with a mean difference of +2.09 mU/L and a standard deviation of 7.67 mU/L. ♦) for LDx/NS2400 and black crosses (+) for the PE/GSP. The results of the 472 samples measured in duplicate on both platforms, are deliberately aligned according to the first measurement on the LDx/NS2400. Therefore, one orange rhomb for sample (n) always overlaps with one orange rhomb from the next sample (n + 1), which gives the impression of a sigmoid line. However, important is, that for each sample there are in total four results plotted on the y-axis . Over the entire range we observed a good agreement between both assays. However, there is one sample pair on the LDx/NS2400 platform , and 15 sample pairs on the PE/GSP platform , that showed a big discrepancy between the two measurements (on the same platform). The reason for this discrepancy is most probably a problem with the washer or disc remover. Both test kits are so called sandwich ELISA test, where the labeled antibodies bind to a TSH molecule, and this complex binds to a second antibody that is immobilized in the microtiter plate. Excess labeled antibodies are then removed during disk removing and washing. In case the disk from the sample collection device is not removed, the respective well is insufficiently washed, and remaining excess label, or the floating disk itself [During the comparison described in chapter 3.1 we observed some very discrepant results, where elevated TSH concentrations could not be reproduced when the measurement was repeated in duplicate on the same platform. Therefore, we compared 472 newborn samples with TSH values between 2 and 460 mU/L in duplicate on both platforms. In k itself  will leaDay-to-day reproducibility of the calibration curves for the PE/GSP platform has been described previously , with CVThe distribution of TSH values was calculated from 748,180 routine newborn samples measured on the PE/GSP platform  and fromn = 260). Negative concentrations are physically impossible, and should principally not be reported from the calculation software that is part of the PerkinElmer calculation software. For the LDx/NS2400 platform the results are much less right skewed, and the calculation software does not calculate negative concentrations. Since TSH values in the low range are already around or below the limit of quantitation, a logarithmic transformation of the TSH values was made [The results from the PE/GSP show a significant right skewed distribution, with a significant number of results with a calculated negative concentration (was made , in ordeQC parameters for the PE/GSP platform have also been described previously, with intra assay CVs of 8.9 and 4.9 and inter assay CVs of 8.1 and 8.3, at 15.0 mU/L, and 59.6 mU/L, respectively . For theIn addition, 36 samples from EQA schemes were measured on the LDx/NS2400 platform. In total, 21 samples were from RfB, 11 from UK-NEQAS and 4 from CDC . QC sampThe two platforms for TSH determination from DBS showed a good correlation, and the most important result is that no misclassifications of confirmed cases with congenital hypothyroidism were observed. Neither on the PE/GSP, nor on the LDx/NS2400 platform. The results of the comparison between PE/GSP and LDx/NS2400 was comparable to the comparison of PE/GSP and PE/AutoDelfia . The samBoth platforms are, without any limitation, suitable for the measurement of TSH from dried blood spots for NBS. TSH values are comparable, and especially in the cut-off range between 10 and 20 mU/L whole blood, there is very good concordance. During the evaluation, and also during routine use of both platforms, no false negative cases occurred. However, due to the narrower distribution of TSH values on the LDx/NS2400 platform, the recall rate, and therefore the number of false positive results was lower.Apart from the TSH test kit performance, the LDx/NS2400 has one major advantage compared to the PE/GSP. The LDx/NS2400 is an “open” instrument, it can be used also for assays, that use organic solvents, which is strictly inhibited on the closed PE/GSP instrument. Furthermore, all components of the LDx/NS2400 can be used in standalone mode; that means, for example, the washer, disc remover and fluorimeter can also be used for other tests. In addition, those components can also be used during downtime of the complete instrument. This makes the LDx/NS2400 platform much more flexible, and for medium size NBS laboratories it offers the possibility total automation with only one automated platform, instead of also investing directly in a second backup instrument.


Supplemental Digital Content is available in the text Melasma is a common acquired hyperpigmentation of the skin. The clinical manifestations are light brown or dark brown patches symmetrically distributed on the cheeks, forehead, and mandibular with different shades and unclear borders. The treatment of melasma is extremely challenging due to its easy relapse and hard to cure. As a classical method of traditional Chinese medicine, acupuncture has been used to treat melasma in clinical practice. However, there is no systematic review and meta-analysis of acupuncture on melasma. This study aims to assess the efficacy of acupuncture for melasma.2 and I2 tests will be utilized to assess the statistical heterogeneity of evidence. We will conduct a meta-analysis if the studies can be combined. Otherwise, a narrative synthesis will be performed. Article screening and data extraction will be independently evaluated by 2 reviewers trained in methodology according to the established selection criteria. Any disagreements between the 2 reviewers will be resolved by reaching a consensus with the other authors (the third reviewer).We will perform a comprehensive retrieval in the following electronic databases: PubMed, EMBASE, the Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure (CNKI), Wan Fang Database, VIP database, Chinese Biomedical Literature Service System (SinoMed), Chinese Biomedicine (CBM) database, and TCM Literature Analysis from inception to July 1, 2021, to identify any eligible study. We include all randomized controlled trials (RCTs) without any limitation of blinding or publication language, and exclude cohort studies and case reports. The meta-analyses will be conducted by using the Stata16. We will use the Cochrane risk of bias tool for randomized trials to assess the risk of bias of included studies. The XThis study will systematically evaluate the clinical efficacy of acupuncture for melasma.The conclusion of this study will provide evidence to ensure the efficacy of acupuncture for melasma, so as to provide an objective and scientific basis for clinical practice.No ethical approval will be required because the data used are not linked to individual patient. The results of this review will be published in a peer-reviewed journal.INPLASY2021110097. Melasma affects especially women during menstruation, especially in thirties and forties Asian women. The incidence rate of Asian women of childbearing age is as high as 30%. There are 3 types of melasma, which are classified according to their distribution on the face, including central facial, cheekbone, and mandibular type.,6 But other sites may also be involved, forming extra-facial melasma. Earlier studies classified melasma as epidermis, dermis, or mixed according to Wood's lamp examination, but studies using laser confocal microscopy showed that all melasma are mixed, indicating that there is a common pathophysiology.,8 Factors affecting melasma may include ultraviolet radiation, genetic factors, hormones, inflammation, reactive oxygen species, melanin synthesis increase, hyperplasia of blood vessels in skin lesions, skin barrier impairment, and basement membrane disruption. In addition, sleep disorder, using inferior cosmetics such as mercury and excessive lead content, exposing to heat radiation such as cooking, thyroid diseases, female reproductive system diseases, and liver diseases can also induce or aggravate melasma. Some studies have also reported melasma in patients following stressful events and affective disorder such as depression.,11 Current treatments for melasma include topical medications, chemical strippers, laser and phototherapy, and systemic medications, options target photoprotection, melanocytes activity, various dermal or epidermal cells that signal to melanocytes, and abnormal tissue changes due to photoaging. Melasma is no longer considered to be a static process, but a complex epidermal–dermal dynamic interaction with various cell types, inflammation, oxidative stress, and photodamage all contribute significantly to this process. Despite a strong demand for treatment, the treatment of melasma remains highly challenging with inconsistent results and almost constant relapses. Chloasma belongs to the category of Chinese medicine “Soot black plaque,” “butterfly plaque,” “liver plaque,” “face dust,” and other diseases. Acupuncture has been used in clinical treatment of melasma, which may be related to acupuncture to dredge meridians, promote blood circulation and remove stasis, and promote circulation of qi and blood.–18 Traditional Chinese medicine believes that the cause of this disease mainly lies in kidney-yang deficiency, liver stagnation and qi stagnation, spleen deficiency, etc. Acupuncture has a better therapeutic effect for this disease with fewer side effects, and is easy to be accepted by patients.,20 According to published articles, there is a lack of high-quality evidence for the treatment of melasma with acupuncture, and no systematic review and meta-analysis have been conducted in this regard. Therefore, we will systematically evaluate the clinical efficacy of acupuncture for melasma, so as to provide an objective and scientific basis for clinical practice.Melasma is a commonly acquired hypermelanosis that affects sun-exposed areas of the skin, with frequent facial involvement.2https://doi.org/10.37766/inplasy2021.11.0097). This protocol has been checked with Preferred Reporting Items for Systematic review and Meta-Analysis Protocols (PRISMA-P) checklist. If the protocol is modified, we will describe the information in the final report.This systematic review protocol has been registered on INPLASY (registration number: INPLASY2021110097), and is available in full on the inplasy.com  of acupuncture for the treatment of melasma without any blinding or publication language restrictions. We will also exclude cohort studies, case reports, and duplicate publications.2.1.2Participants will include patients diagnosed with melasma based on medical history and typical clinical manifestations: there are no restrictions on the age, gender, or race of the subject.2.22.2.1We will include all RCTs in which the treatment group uses acupuncture alone. Acupuncture treatment is defined as acupuncture at acupoints on the meridian, including manual acupuncture or electroacupuncture, but not other acupuncture therapies, such as ear acupuncture, scalp acupuncture, dry acupuncture, and acupressure therapy.2.2.2The control group uses only conventional treatment. Conventional treatments include topical medications, chemical stripping agents, laser and light therapy, systemic medications, and other necessary treatments.2.32.3.1 quantification is carried out according to the area, color depth, and color uniformity of melasma. Pigmentation area assessment: forehead (F), right cheek (MR), left cheek (ML) and lower jaw (C) are divided into 4 areas, with weights of 30%, 30%, 30%, and 10%, respectively. The color depth (D) and uniformity (H) scores are counted as 0 to 4 points: 0 means nothing, 1 means slight, 2 means moderate, 3 means obvious, and 4 means maximum. MASI = forehead [0.3A (D + H)] + right cheek [0.3A (D + H)] + left cheek [0.3A (D + H)] + lower jaw [0.1A (D + H)]. The maximum is 48 points, the minimum is 0.The main outcomes include Melasma Area and Severity Index (MASI),2.3.2 VISIA image analysis, noninvasive physiological function test, reflectance confocal microscopy (RCM), dermoscopic observation and evaluation of the improvement of the number and shape of blood vessels in the skin lesions before and after treatment of melasma, Physician's Global Assessment (PGA), patient satisfaction evaluation, safety indicators, and the number of adverse events.Secondary results include scanning reflectance spectrophotometer detection technology (colorimetric method),2.42.4.1http://links.lww.com/MD2/A765.We will search PubMed, EMBASE, the Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure (CNKI), Wan Fang Database, VIP database, Chinese Biomedical Literature Service System (SinoMed), Chinese Biomedicine (CBM) database and TCM Literature Analysis and Retrieval Database from inception to July 1, 2021, to identify any eligible study. We include all RCTs without any limitation of blinding or publication language, exclude cohort studies and case reports. Detailed search strategy will be shown in Appendix 1, 2.4.2We will search the US National Institutes of Health Ongoing Trials Register, the WHO International Clinical Trials Registry Platform, Chinese Clinical Trial Registry (ChiCTR), ClinicalTrials.gov, Google Scholar, and Baidu scholar for any relevant ongoing or unpublished trials.2.52.5.1The titles and abstracts of all searched studies will be independently evaluated by 2 reviewers (CZ and HY) trained in methodology according to the established selection criteria. Any disagreements between the 2 reviewers will be resolved by reaching a consensus with the third reviewer (QT). If necessary, they will read the full text of all included studies. A PRISMA flowchart will be drawn to illustrate the study selection process Fig. .2.5.2Citations will be independently screened by the 2 authors (CZ and HY), and data will be extracted using a standardized data extraction table. The following information will be determined for each trial: authors name, publication year, inclusion and exclusion criteria, number of patients and reviews, type of acupuncture, and outcome measures. Any disagreement will be resolved by consensus or consultation with a third review author.2.5.3Two reviewers (CZ and HY) will independently assess the risk of bias using the Cochrane Risk of Bias Tool of Randomized Trials. They will compare their own assessments and discuss any differences of opinion between them. If they fail to do so, they will be arbitrated by a third-party reviewer (QT). Area to be evaluated: Is there enough sequence generation (selection bias)? Is the allocation sufficiently masked (selection bias)? During the course of the research, is there sufficient knowledge about the interventions allocated? Participants and personnel (performance bias), result assessor (detection bias); are incomplete result data adequately resolved (attrition bias)? Is there no hint of selective result reporting (reporting bias) in the research report? Is this study apparently free of other issues that might put it at risk of bias? On the basis of the relevant information extracted from each qualified study, the risk of bias in each area will be divided into high risk, low risk, and unclear risk of bias.2.5.4We will conduct a meta-analysis if the studies can be combined. For dichotomous variables, a risk ratio (RR) with 95% confidence interval (95% CI) will be used for analysis. Standardized mean difference (MD) with 95% CI will be calculated for continuous data.2.5.5We will attempt to contact the original researchers via email to obtain any missing or inadequate data. Then, we will perform a sensitivity analysis using imputations of missing outcome data of dichotomous outcomes in best-worse and worse-best case scenarios to assess the potential impact of loss to follow-up. If we cannot collect accurate data, we will exclude these studies.2.5.62 and I2 tests will be utilized to assess the statistical heterogeneity of evidence. When P ≥ .1 and I2 ≤ 50%, it is considered that there is no statistical heterogeneity or the heterogeneity is small. When P < .1 and I2 > 50%, the result indicates that there is a statistical heterogeneity. We will calculate the I2 statistic . We will use subgroup analysis to explore the causes of heterogeneity among the results of studies.The X2.5.7If more than 10 articles are included, we will use funnel plot and Egger test to assess publication bias. If there is publication bias, we will use the clipping-compensation method to further evaluate the impact of publication bias on the results. If the impact is not large, the authenticity of the results is better, and if the impact is relatively large , it will be fully included in the results to discuss the impact of publication bias on the results.2.5.8If there is no heterogeneity, use a fixed-effects model to synthesize the data; if there is significant heterogeneity, use a random-effects model for analysis. We will provide a narrative synthesis of the outcomes and results of the studies if a meta- analysis is not possible.2.5.9If a sufficient number of RCTs are included in the review, we plan to conduct subgroup analysis to explore the source of heterogeneity. The subgroup analysis will be based on the type of acupuncture  and the test time of the secondary results (3 or 7 days after the intervention).2.5.10We plan to use the “leave-one-out” methods to conduct sensitivity analyses for the main outcomes to confirm the reliability of our findings.2.5.11 The GRADE approach evaluates the quality of evidence as “high,” “moderate,” “low,” or “very low” by the outcome. The quality of evidence can be reduced by 5 factors  and increase by 3 factors . The author will independently reduce or improve the quality of evidence and resolved disagreements by discussion.The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method will be used to summarize the quality of evidence and provide a “Summary of Survey Results” table.2.5.12We will provide the date of any amendment, a description of the change and the rationale in the event of protocol amendments.2.5.13Patient and public were not involved in this study.2.5.14No ethical approval will be required because the data used are not linked to individual patient. The results of this review will be published in a peer-reviewed journal.3 Melasma has a significant impact on appearance, causing psychosocial and emotional distress, and reducing the quality of life of the affected patients. Patients commonly report feelings of shame, low self-esteem, listlessness, dissatisfaction, and the lack of motivation to go out, Suicidal ideas have also been reported in the literature. The mechanism of acupuncture treatment of melasma is still in its infancy and needs to be further developed. Preliminary studies have found that balancing oxidation and antioxidant functions, improving blood rheology, and regulating sex hormones may be an important mechanism of acupuncture treatment of melasma. We hope to evaluate the effectiveness of acupuncture in the treatment of melasma from published RCTs. The results of this review will help clinicians use acupuncture in the treatment of melasma.Treatment of melasma is challenging, usually difficult to treat, and often recurs.Conceptualization: Liheng Tang.Data curation: Changyun Zhang, Huijuan Yu, Qiwen Tan.Formal analysis: Changyun Zhang, Huijuan Yu, Qiwen Tan.Methodology: Jin Xian.Project administration: Xin Zhang.Resources: Jin Xian, Ye Zhang.Software: Liheng Tang, Jin Xian.Supervision: Qiwen Tan, Xin Zhang.Visualization: Liheng Tang, Jin Xian, Ye Zhang.Writing – original draft: Liheng Tang.Writing – review & editing: Ye Zhang, Xin Zhang.


Musculoskeletal injuries occur frequently after road traffic crashes (RTCs), and the effect on work participation is not fully understood. The primary aim of this review was to determine the impact of sustaining a musculoskeletal injury during an RTC on the rate of return to work (RTW), sick leave, and other work outcomes. The secondary aim was to determine factors associated with these work-related outcomes. An electronic search of relevant databases to identify observational studies related to work and employment, RTC, and musculoskeletal injuries was conducted. Where possible, outcome data were pooled by follow-up period to answer the primary aim. Fifty-three studies were included in this review, of which 28 were included in meta-analyses. The pooled rate of RTW was 70% at 1 month, 67% at 3 months, 76% at 6 months, 83% at 12 months, and 70% at 24 months. Twenty-seven percent of participants took some sick leave by one month follow-up, 13% by 3 months, 23% by 6 months, 36% by 12 months, and 22% by 24 months. Most of the factors identified as associated with work outcomes were health-related, with some evidence also for sociodemographic factors. While 70% of people with RTC-related musculoskeletal injury RTW shortly after accident, many still have not RTW two years later. Musculoskeletal injuries occur frequently after road traffic crashes (RTCs) to varying levels of severity. This includes whiplash injury, which is one of the most common injuries ,2,3, aloWork is important for many reasons, including the promotion of positive health outcomes. There is a recognized link between harm to mental and physical health, and prolonged time off work ,9,10. ThRecent work in the field of orthopaedic trauma (not specific to RTCs) suggests that 61% of injured persons RTW within 2 years post-injury  with an Delayed RTW and absence from work are not the only work-related outcomes that could be affected by an RTC-related musculoskeletal injury. Work capacity, work ability, and health-related work productivity loss are also key outcomes to consider. Work capacity is often considered to be an individual’s capacity to meet the demands of their job, and may require modification of duties or hours of work for example. Work capacity is an important construct when considering the outcomes of RTC, as medical clearance to RTW is often required either by the employer or the insurer . Work abThe primary aim of this systematic review is to determine the impact of sustaining a musculoskeletal injury during an RTC on five work-related outcomes: the rate of RTW following injury, the utilisation of sick leave, work capacity, work ability, and health-related work productivity loss. The secondary aim is to determine factors associated with these work-related outcomes in people with musculoskeletal injuries as a result of an RTC.This review was conducted and reported in compliance with the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) . This syAn electronic search was conducted in PubMed, EMBASE, Cumulated Index to Nursing and Allied Health Literature (CINAHL), Web of Science, PsycINFO, and Australian Transport Index databases on 2 July 2020. To identify relevant articles, a combination of search terms was used related to work and employment, RTC, observational study design, and musculoskeletal injuries . The seaThis systematic review included published studies of individuals who sustained musculoskeletal injuries during an RTC on work-related outcomes.Studies were eligible for inclusion if participants were adults who sustained a musculoskeletal injury during an RTC. Studies that investigated outcomes after neurological injuries such as spinal cord injury and traumatic brain injury were excluded, as recent work has already been done in this space ,28. OtheAdults were the focus of this review, and thus studies with exclusively paediatric cohorts were excluded. However, studies that consisted of a small percentage of adolescent participants were still included if adults remained the majority of the cohort ,30,31,32Five work-related outcomes were the focus of this review, and included studies needed to report at least one of: (i) RTW rate, (ii) sick leave, (iii) work ability, (iv) work capacity, or (v) health-related work productivity loss. These work-related outcomes could be reported as either a primary or secondary outcome of the study.Included studies were observational in design, including cross-sectional, retrospective, and prospective. If participants engaged in rehabilitation programs that were considered ‘usual care’ or those that occur in ‘real world’ settings , those studies were considered observational and were included in the present review. Studies were excluded if they were randomized controlled trials or case studies, or pre-post-intervention designs implemented by the study authors . These criteria were applied in order to capture outcomes from participants recovering under ‘real world’ circumstances, and not experimentally controlled care.Only studies published in English were eligible for this review.Title and abstract screening and full-text screening was conducted independently by two researchers  in Microsoft Excel. Author EG screened all title and abstracts, with the second screening conducted by TA (60%), CB (30%) and ES (10%), allocated at random. Full-text screening was equally distributed between EG, TA and ES. Allocation to author for screening occurred in a manner to enable equal crossover amongst the three authors . Discrepancies were resolved by group discussion or by senior author VJ. Reference lists of included studies and relevant systematic reviews identified in the search were reviewed for eligible studies.Data extraction was divided evenly amongst the authors  so that each study was examined independently by two authors, with consensus achieved by consultation with the senior author VJ. Allocation for data extraction was similar to full-text review in that there was approximately equal cross-over amongst authors. Data were extracted into a predetermined Excel form that has been described in detail previously . AuthorsStudy quality was assessed with the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) Study Quality Assessment Tools for observational cohort and cross-sectional studies, and case–control studies . Co-authInter-rater reliability of the quality assessment tool was examined using kappa (κ) statistics . Reliability was considered as slight (0.00–0.2), fair (0.21–0.4), moderate (0.41–0.6), substantial (0.61–0.8) or almost perfect (0.81–1.0) .2 = 25% was considered low, 50% moderate, and 75% high [Meta-analysis was conducted provided that at least two studies reported on the same work outcome including variability estimates. Categorical outcomes  were transformed using the Freeman-Tukey double arcsine method  and pool75% high .Sensitivity analyses were performed to assess the robustness of the outcomes by excluding the studies rated as ‘poor’ on the quality appraisal tool. Narrative synthesis was used to summarise and explain the conclusions across the studies.n = 10) used  the same participant cohorts [ cohorts ,42,43,44 cohorts ,39,40,43 cohorts ,42, the  cohorts ,39,42,44n = 22, 42%), followed by Australia , North America , South America , Africa  and the Middle East . Participants were sampled through various means including insurance companies and the general public , various musculoskeletal injuries  , and mixed cohorts consisting of whiplash and other musculoskeletal injuries . The follow-up assessments from time of injury ranged from 3 days [Of the m 3 days  to 8.5 ym 3 days .Four studies were included in which adolescents were included in the study cohort ,30,31,32p < 0.001) with 602/742 agreements [Inter-rater agreement for quality appraisal of individual studies was substantial , clearly specified and defined the study population , selected or recruited participants from same or similar populations during the same time period , and had a timeframe that was sufficient to reasonably expect to see an association between exposure and outcome  .Results of the quality appraisal of the individual studies are presented in 43, 81%) . Howevern = 53), outcomes related to RTW were reported by 27 studies , sick leave by 23 studies (43%), work capacity by 13 studies (25%), work ability by 6 studies (11%) and productivity loss by 4 studies (8%)  . Pooled 2 99%; 4 studies) up to 1 month, 67%  up to 3 months, 76%  up to 6 months, 83%  at 12 months and 70%  at 24 months  of injured workers had used sick leave at 1 month post-RTC, 13%  at 3 months, 23%  at 6 months, 26%  at 12 months and 22%  at 24 months , between 2 and 4 weeks by 12% , and sick leave of more than 4 weeks was reported by 15%   . InjuredEleven studies (thirteen publications) reported outcomes related to modifying duties at work and/or the inability to work  for timeSix studies ,71,75,79n = 38)  was 15% at 12 months in one study . When ren = 38) . BunketoHoving and colleagues (2003)  reportedn = 13/15 participants with facial injuries [p = 0.96) of not filing for an economic loss claim  compared with those who had musculoskeletal injury only  [Four studies reported measures of health-related productivity loss ,58,68,72injuries . Those wp < 0.01) (p < 0.001) (p < 0.001) and 3 months   for 1, 3, 6, and 24 months but not for 12 months  . Sick le< 0.001) . Sick le< 0.001) . Three on = 30/53) reported information on  factors associated with work-related outcomes (n = 14/27) investigated associated factors. For sick leave, 39% (n = 9/23) investigated associated factors, for work capacity 23% (n = 3/13), for work ability 50% (n = 3/6), and for productivity loss 75% (n = 3/4) of studies investigated associated factors , greater severity of injury, being admitted to hospital, having a higher baseline OMPSQ score, having lower baseline mental health-related quality of life, lower pre-injury fitness levels and the presence of a pre-injury chronic illness ,32,42,57p < 0.05) [Two studies reported associations between taking time off work/sick leave and being female ,54 and t < 0.05) . No sign < 0.05)  with age < 0.05) .Four studies investigated the relationship between injury-related factors and sick leave. Sustaining a whiplash injury was a risk factor for taking more sick leave  and not One study reported that those who were unable to continue in their pre-injury work capacity scored significantly worse on all health outcome measures including the SF-36 and pain catastrophizing scale , whereasp = 0.12) [One study reported no evidence of an association between gender and status as work disabled ( = 0.12) ). As rep = 0.12) , reducedp = 0.024). An economic analysis of data from Denmark found those with WAD injuries who were awarded compensation on the basis of injury severity were more likely to have lost earning capacity in the long term [Barbosa et al.  reportedong term . O’Hara ong term  reportedn = 498 total from the age of 17 y and above, with a mean (SD) age of 40.2 (16) y for neck injuries, 35.7 (17) y for lower back injuries, and 38.9 (15) for lower limb injuries. Any 17 y old participants would have been more than 1 standard deviation outside of the mean. In a normally distributed dataset, 68% of the cohort is within one standard deviation of the mean (mean +/−1 SD).As described in the methods The potential impact of including these four studies on the pooled meta-analyses is minimal. Only one study  contribuThis systematic review identified 53 observational studies in which RTW, sick leave, work capacity, work ability, and health-related work productivity loss were measured in adults with a musculoskeletal injury after RTCs. Findings from the meta-analyses within this review should be interpreted with caution, as the degree of statistical heterogeneity was high. Each pooled statistic was the product of the combination of different studies, and represents a combined estimate based on similar data drawn from at least two studies. Outputs from those meta-analyses demonstrated the pooled RTW rate generally increases post-injury from 70% of injured persons having RTW at 1 month, to 67% at 3 months, 76% at 6 months, and 83% at 12 months. These findings suggest that most injured persons are back at work within a month of their RTC, and of those who are not, some go on to return gradually to work over the first year after RTC, whilst some will not have a successful RTW at 1 year post-injury. Approximately one-third of injured workers used sick leave in the year after RTC, and approximately one-sixth used more than 4 weeks of sick leave. Using sick leave may be the best (or only) option for individuals without suitable modified duties available to them in their workplace. Results of four studies demonstrated a varying percentage  of workers used modified duties at work. Few studies identified by this review measured work ability  or health-related work productivity loss . More research is needed in this client population regarding these outcome measures for appropriate conclusions to be drawn.a priori sample size calculations in observational studies has implications for detecting a difference in the primary outcome between sub-groups. In addition, many studies did not provide an indication of how representative their sample was of the true population of those injured by RTCs by omitting a comparison between those who consented versus those who declined to participate. However, the use of data such as the age and sex of those who declined to participate may not have been available to researchers, as there are ethical concerns related to the collection of data from those who have not consented.There are several key aspects of the methodology of the included studies that are worth highlighting, and should be considered with the findings. It is encouraging to have a high percentage of prospective observational studies within this review, and many with large sample sizes from administrative databases, which can both be considered as markers of methodological quality within the field. On the other hand, administrative databases may be biased representations of the total population of adults with musculoskeletal injuries after RTC, over-emphasising the experience of those who presented to hospital  or those who were eligible for compensation (insurance databases), depending on the jurisdiction in which they live. Regarding type of injury, the most common injury type were whiplash injuries. This fits with the frequency of whiplash injuries in RTC and the emphasis placed on these injuries in the literature ,85,86,87Of the studies reporting RTW rates, the most data were available for 12 months post-injury, demonstrating that 83% of adults with a musculoskeletal injury after RTC had returned to work by 12 months. RTW rates range from 1 month (69.5%) to 12 months (83%) and suggests that those who have not returned by 1 month may take several months to achieve a successful RTW. Vocational rehabilitation programs can facilitate RTW by optimising work participation usually with a combination of medical, psychological, social and/or occupational strategies: for example, assessments of functional capacity and vocational goals, vocational counselling, training and work experience opportunities (particularly within the workplace), and assistance with job seeking . FindingExisting evidence for vocational rehabilitation, and rehabilitation more broadly, also needs to expand to encompass developing nations. The World Health Organization’s ‘Decade of Action for Road Safety 2021–2030′ calls for the provision of rehabilitation to all RTC injured persons, and particularly for “protections for people with disabilities to keep their jobs or be hired in new jobs through the provision of incentives for employers will further alleviate the socioeconomic consequences of permanent disability”. LiabiliInterestingly, the pooled data (5 studies) for 24 months post-injury resulted in the same RTW rate as one month post-injury—70%. This could reflect a portion of injured persons being unable to sustain their RTW, which is known to occur . This coInconsistency in the definition of RTW, with no universally accepted standard, makes it difficult to carry conclusions across different types and mechanisms of injuries. In this review, some studies considered return to exactly the same job, hours and/or conditions e.g., ,57,62), ,62, 57,6Meta-analyses of sick leave data demonstrated approximately 1 in 4 injured persons used sick leave in the first year after their RTC, and for those who did use sick leave, 15% used it for more than 4 weeks. Musculoskeletal disorders in general are responsible for a significant amount of sickness absence and work disability, with the associated loss in productivity being equivalent to an estimated 2% of the European Union’s gross domestic product . In a SwA wide range of domains were assessed and found to be associated with the work-related outcomes within this review. Categories of independent variables included sociodemographic factors, psychosocial health, injury and RTC characteristics, pre-crash mental and physical health, educational attainment, employer support, and type of work. The significance of any association, and if present, the direction of the association varied for many of these characteristics across multiple work-related outcomes. There are some links that could be drawn from the results of the present review; for example, people who were married, divorced or widowed were more likely to have longer sick leave (vs single people)  and a deThis review has a number of strengths. A thorough database search was conducted across five scholarly databases and one grey literature source specific to the transport industry. Independent review by two co-authors for screening, data extraction and appraisal led to high ratings of agreement. Results were presented by timeframe, to aid in the interpretation of recovery after RTC over time. Finally, five work-related outcomes were investigated, expanding the knowledge base beyond a focus on achievement of RTW.There are also limitations that must be acknowledged. There were high levels of heterogeneity in the results of the meta-analyses. The database search was restricted to studies published in English which may have contributed to the under-representation of results from developing countries. There are a number of validated tools for measuring work capacity and work ability that this review was expecting to find . HoweverThis review found that the pooled RTW rate increases in the first-year post-injury, from 70% at 1 month to 83% at 12 months. Approximately one-third of injured workers use sick leave in the year after RTC, and approximately one-sixth used more than 4 weeks of sick leave, reflecting a significant interruption to their employment and cost to their employer. Less than half of injured persons used modified duties at work following RTC-related musculoskeletal injury. More research is needed to understand the impact of RTC on work ability and health-related productivity loss. Clinicians are encouraged to consider the multiple potential factors of influence  for a client’s recovery and RTW after RTC, and be familiar with the legal and insurance frameworks operating in their jurisdictions.


The TRIPOD 29-item checklist was used to evaluate the adherence to reporting guidelines of the studies, and the adherence rate to each item was computed. We conducted a meta-analysis to determine the pooled sensitivity and specificity of machine learning models for detecting KC. (3) Results: Thirty-five studies were included in this review. Thirty studies evaluated machine learning models for detecting KC eyes from controls and 14 studies evaluated machine learning models for detecting early KC eyes from controls. The pooled sensitivity for detecting KC was 0.970 (95% CI 0.949–0.982), with a pooled specificity of 0.985 (95% CI 0.971–0.993), whereas the pooled sensitivity of detecting early KC was 0.882 (95% CI 0.822–0.923), with a pooled specificity of 0.947 (95% CI 0.914–0.967). Between 3% and 48% of TRIPOD items were adhered to in studies, and the average (median) adherence rate for a single TRIPOD item was 23% across all studies. (4) Conclusions: Application of machine learning model has the potential to make the diagnosis and monitoring of KC more efficient, resulting in reduced vision loss to the patients. This review provides current information on the machine learning models that have been developed for detecting KC and early KC. Presently, the machine learning models performed poorly in identifying early KC from control eyes and many of these research studies did not follow established reporting standards, thus resulting in the failure of these clinical translation of these machine learning models. We present possible approaches for future studies for improvement in studies related to both KC and early KC models to more efficiently and widely utilize machine learning models for diagnostic process. Corneal diseases are the second largest cause of blindness worldwide, behind only cataract in overall importance . KeratocIn the medical field, the recent availability of biomedical data has led to the advent of the big data era ,7, creatIncreased detailed data about the cornea resulting from corneal topography and tomography systems are essential for diagnosing KC. They are especially useful for detecting early indications of KC, prior to the development of typical KC clinical characteristics . CornealThere is no existing study that summarizes the use of machine learning in KC, identifies limitations, and makes recommendations for future directions. The aim of this study and meta-analysis was to systematically review all currently available literature to determine accuracy (through the use of pooled sensitivity and specificity) of machine learning in the detection of KC, addressing this knowledge gap.A web-based systematic literature search was performed for articles published from inception through 28 February 2021, on PubMed, Web of Science, and MEDLINE . DatabasPubMed, Web of Science, and MEDLINE were interrogated using search strings pertaining to keratoconus and machine learning. Key words were used by search engine and designated filters according to PRISMA  guidelines . The seaThe following sections define the inclusion and exclusion criteria.-Full text original papers that evaluated machine learning algorithms in the diagnosis of KC;-No limit on the year of publication was applied;-Publications only in the English language were included;-Publications in which KC was the only corneal condition evaluated.-Criteria for exclusion:-Publications evaluating other corneal diseases without focusing only on KC;-Publications evaluating the efficacy of machine learning in treating KC (treatments);-Review papers;-Publications in which no machine learning algorithms were included, but only statistical research was undertaken;-Non-English publications.Primary research articles, meeting the following requirements were deemed suitable for inclusion in this review:The review articles were imported into Endnote (version X9), which was used to perform the initial screening. Two independent reviewers (K.C. and S.S.) screened the titles and abstracts of journals for possible qualifying studies, and inconsistencies were settled by consensus. Both reviewers thoroughly analyzed all potential qualifying studies for inclusion/exclusion. .A customized analysis form was generated in Microsoft Excel. The following items were reviewed from each study: authors, publication year, country, study objective, sample size, reference standard diagnosis methods, corneal imaging systems used to generate data and machine learning method used, indicators of studies’ quality, and the number of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN). If the number of TP, FP, TN, or FN were not presented, then these values were derived from the data provided, such as sensitivity and specificity. The meta-analysis omitted studies that lacked data on TP, FP, TN, or FN or that lacked the capacity to measure these data to create a two-way contingency chart.www.tripod-statement.org (accessed on 7 June 2021)) statement relevant to model development. This statement contains a 20-item checklist, totaling 31 items when all sub-items are included. The checklist includes questions about the title, abstract, background, methods, results, discussion, supplementary material, and funding information. Two items  were omitted since they were irrelevant to the research covered in this review. Each study was therefore evaluated on a total of either 28 or 29 possible items. This total number of items varied between 28 and 29 since item 14b  is reported, score not applicable”) may be rated as “not applicable” and was thus omitted from the calculation of reporting adherence. If a study had data for several models, we extracted data for the model with the highest performance. Each included item received a score of “1” for adherence and a score of “0” for non-adherence. Multiple items  in the TRIPOD analysis were derived from several sub-items (the sub-items for each number can be found in www.tripod-statement.org (accessed on 7 June 2021)). The score was therefore determined by the combination of several elements rather than a single element. The results of each TRIPOD item for each paper and the level of reporting adherence for each TRIPOD item were documented systematically in a spreadsheet.We evaluated the reporting completeness of machine learning research in this study by referring to the TRIPOD  (Version 1.3.1056-1) for Windows. To measure the overall machine learning performance for KC detection, the sensitivity and specificity values for all presented models were pooled, following the bivariate meta-analysis method of Reitsma et al.  using liA hierarchical summary receiver-operating characteristic (HSROC) curve was fitted. Each individual study was presented as a circle and plotted within the HSROC curve. The summary point was represented by a dot surrounded by a 95% confidence interval (95% CI).The primary outcome indicator was the diagnostic accuracy of machine learning algorithms through a variety of imaging technologies for the identification of KC, as determined by the pooled sensitivity and specificity values. Of the initial literature search, 532 studies were retrieved, and 280 duplicates were omitted. Following review of the title, abstract, and full text, 35 studies were included in the review. There was an increasing trend of machine learning studies in KC published over time , with thThe 35 articles on machine learning and KC were reviewed were classified into three categories based on their aims: detecting KC eyes from controls, differentiating early KC from controls, and identifying different KC severities. Each study focused on one or more of these aims, with 12 research papers focused on KC versus control, 4 research papers focused on early KC versus control, 1 article focused on KC severity, and 18 publications with multiple aims. There was no study examining the progression of KC.Following classification of studies with multiple aims, the following results were obtained: 30 studies ,42,43,44n = 4) [n = 3) [n = 2) [n = 1) [n = 1) [n = 2) [n = 3) [n = 2) [n = 1) [n = 1) (conducted by our group [n = 3) [n = 1) [n = 2) [Of the 35 studies, 33 focused on developing machine learning models, while 2 ,46 involn = 4) ,17,18,30 [n = 3) ,42,49, F [n = 2) ,23, the  [n = 1) , India ( [n = 1) , Brazil  [n = 2) ,45, Belg [n = 3) ,33,37, H [n = 2) ,33, Aust [n = 1) , Australur group ), Spain  [n = 3) ,40, Taiw [n = 1) , and Chi [n = 2) ,47, and  [n = 2) ,29,31,34n = 23) employed a single machine learning algorithm, while 4 studies [A total of 26 studies ,38,39,40 studies ,29,31,38Four articles directly analyzed images ,42,43,44p = 0.91).Meta-analysis was performed on 26 of the 30 studies, with 4 studies ,39,41,43The pooled sensitivity and specificity for KC versus control were 0.970 (95% CI 0.949–0.982) and 0.985 (95% CI 0.971–0.993). Pooled performance was computed using a bivariate random-effects model and represents a summary estimate of the sensitivity and specificity values  obtained from each individual research . The mosA total of 13 studies ,46,47,48p = 0.18).Ten studies were included in the meta-analysis, out of a total of fourteen. As shown by an asterisk (*) in Overall, the pooled sensitivity was 0.882 (95% CI 0.822–0.923) and pooled specificity was 0.947 (95% CI 0.914–0.967) for early KC versus control. The most widely used imaging machines in the detection of early KC from control eyes were the Pentacam (including Pentacam HR) and Orbscan (including Orbscan II and Orbscan IIz). Six studies used Pentacam data, two studies used Orbscan data, and an additional two studies used data from either GALILEI, or a combination of Pentacam and Corvis. Early KC detection  was corrThe pooled diagnostic performance of detecting KC compared to controls was superior to that of early KC with sensitivity (0.970 (95% CI 0.949–0.982) vs. 0.882 (95% CI 0.822–0.923) and specificity (0.985 (95% CI 0.971–0.993) vs. 0.947 (95% CI 0.914–0.967)). This difference implied that early KC detection using machine learning algorithms are still in their infancy.The Pentacam tomography system was the most commonly used corneal imaging device for both the KC and early KC categories. In Along with detecting KC eyes as a distinct category, six of the published studies grouped their KC eyes into clinical stages and used machine learning algorithms to identify each stage separately. These studies classified KC eyes into various categories based on a variety of measures. Kamiya et al.  classifiThe first attempt at proposing a data-driven KC classification scheme was based on density-based clustering by Yousefi et al.  using OCPublications had only low to moderate adherence to TRIPOD items, ranging between 3% and 48%, with a median of 28%. Each item on the TRIPOD checklist was adhered to by between 0% and 69% of studies, with an average (median) of 23% . Eight iThis is the first comprehensive meta-analysis on KC and machine learning, and it has demonstrated that by using data from a variety of corneal imaging devices, machine learning can reliably distinguish KC eyes from control eyes (pooled sensitivity > 0.90). However, the performance of machine learning models in distinguishing early KC eyes from controls was poorer, with the maximum pooled sensitivity of 0.88. Overall completeness of model was evaluated using the TRIPOD guidelines, and standard reporting compliance was found to be inadequate in all published KC machine learning research so for undertaken.Machine learning models in KC were developed for a variety of imaging systems. Models themselves are therefore not directly interchangeable owing to the different input expectations. This may have a detrimental effect on the clinical translation of these models. For example, Smadja et al. developed a machine learning model that had a sensitivity of 93.6% and a specificity of 97.2% for discriminating normal eyes from early KC in their study . This moThe machine learning models that used data from the Pentacam demonstrated a higher pooled sensitivity and specificity in detecting KC and early KC from control eyes compared to other imaging machines. This is likely due to the ability of the Pentacam machine to generate a wider spectrum of data than other systems, including data on the front cornea, the back cornea, corneal pachymetry, and other areas of the anterior eye segment ,55. A review of the literature on the application of AI to evaluate corneal topography for the diagnosis and early detection of corneal ectasias was recently published . That arOnly when all model components are completely and transparently reported can the model’s potential clinical usefulness be appropriately assessed. The main objective of machine learning models is to help clinicians in making medical decisions about an individual patient . Users oAttempting to replicate the process of model construction using one’s own data, machine learning researchers working in KC will need to specify information on the approach used to handle any missing data, the full prediction model, and a comprehensive explanation of all model-building procedures. However, only one study (3%) defined the whole model-building process , 14% proOne factor contributing to the low percentage of adherence is that not all elements are relevant to all machine learning research. For example, it is impractical to mathematically specify the final model in a study using non-regression techniques such as random forest, support vector machine, or neural network. While this is a legitimate rationale, it is important to refer to relevant TRIPOD items and provide as much information about model creation as possible in order to guide the modelling choices and facilitate subsequent validation.For the studies that reported the number of parameters employed, the range was between 5 and 443. Ruiz et al. used 22 Pentacam parameters to construct a machine learning model for identifying KC and early KC . The parWhile the majority of research developed novel models for KC detection, only four ,22,31,37The development of KC is frequently manifested not only by corneal changes, but also by clinical symptoms such as vision, refraction, and slit-lamp findings. No study has ever incorporated all pertinent data ,47,48,49The bulk of reported studies of KC have used data derived from a single corneal topography or tomography imaging device to train their machine learning models. Thus, there is a dearth of information as to what impact combining data from multiple devices would have on machine learning models in the detection of KC. Other forms of data, such as from the corneal epithelial thickness map produced with optical coherence tomography (OCT) and corneal biomechanical measurements, are also increasingly being recognized as crucial in the diagnosis of KC , particuThe majority of studies employed a specific machine learning method; although, there was limited information on how or why the authors chose a particular algorithm from a large number of potential alternatives. Given that machine learning algorithms are sometimes referred to as ‘black boxes’, it would be advantageous to evaluate several machine learning algorithms on the same data set and select the best one . This prImbalanced sample sizes were found in about half of the studies analyzed ,33,44,46There have been fewer studies on identifying the different stages of KC, and a standardized classification system for KC has yet to be devised. There has been no machine learning study using longitudinal data on KC progression. While applications of machine learning have benefited in predicting and detecting progression indicators in AMD, diabetic retinopathy, and glaucoma ,62,63,64Machine learning has been increasingly used in KC over the last three decades, mostly for the identification of KC and early KC. The advantage of machine learning is that it allows consistent and unbiased diagnosis, which is critical when diagnosing patients at an early stage, as early intervention using treatments such as corneal crosslinking (CXL), could delay or slow disease progression, thus preventing the need for a possible corneal transplant.There is still room to improve the efficiency of machine learning models in detecting early KC. This may be accomplished by allowing the use of all publicly available data, including complete databases from corneal imaging systems, clinical data, genetic data, and other risk factors. There is also a need to maximize the potential of machine learning techniques by optimizing their output at the methodology and data space levels.Currently, there are no successful examples of machine learning models being used in clinical practice. This may be due to a lack of large patient populations to validate results, the utilization of various imaging devices, a local participant group comprised of individuals of various ethnic backgrounds, clinicians’ overall acceptance of machine learning techniques for diagnosis and their relative reliability to humans. External model validation on a diverse patient population, as well as the creation of platform-independent models that can be generalized through several corneal imaging systems, are therefore needed. Finally, research on machine learning in KC should also address additional research gaps in the area, such as classifying KC severity, and identifying and forecasting KC progression.We present an up to date, comprehensive review on the use of machine learning in KC detection and identify the substantial limitations that need to be overcome to make the diagnostic process more efficient for early keratoconus. In light of our findings on pooling detection performance and low adherence to the TRIPOD checklist, we believe that both improved machine learning model performance in early KC detection and improved quality machine learning research in KC is sorely needed. Despite various challenges, the future of integrating machine learning technologies into clinical practice is promising with the advent of advanced imaging modalities. Machine learning can be further investigated for broad application to the entire process of KC detection and management. In particular, some open avenues for research include early KC detection, risk factor evaluation, prediction of progression, and clinical management guidance. However, global collaboration is essential to obtain larger data sets and more robust models.


Currently, neoadjuvant chemoradiotherapy, followed by surgery, is the standard treatment for locally advanced rectal cancer. The use of induction chemotherapy for this tumor is controversial. In this study, the benefits and side effects of induction chemotherapy in locally advanced rectal cancer are evaluated.2 every 3 weeks and capecitabine 1000 mg/m2 twice a day for 14 days every 3 weeks for 2 courses). Then, neoadjuvant chemoradiotherapy  was administered. After 4 weeks, computed tomography (CT) scan of thorax, pelvis, and abdomen with and without contrast was performed. Total mesorectal surgery was performed 6-8 weeks after the end of radiotherapy. Four courses of adjuvant chemotherapy were applied. Pathologic complete response (pCR), margin, sphincter preservation, and adverse effects were assessed.Twenty-nine patients with locally advanced rectal cancer in 2018-2019 were enrolled in this study. Initially, they underwent induction chemotherapy  patients. R0 resection was done in 96.05%. Sphincter was preserved in 44.4% of lower rectal tumors. Two patients (6.9%) did not complete adjuvant treatment. Grade 3 adverse effects were documented in 13.7% of cases during induction chemotherapy and 17.2% of cases during neoadjuvant chemoradiation. Mortality was not reported. Induction chemotherapy, followed by neoadjuvant chemoradiotherapy and surgery, would be an effective and safe modality in locally advanced rectal cancer. Colorectal cancer (CRC) is currently the third leading cause of cancer death in the world, and its prevalence is increasing in developing countries. CRC is more common in men than in women. The global age-standardized incidence rate of CRC is 19.7 per 100 000 persons, in males is 23.6, and in females is 16.3 (The use of neoadjuvant fluorouracil (FU)-based chemoradiotherapy, followed by complete mesorectal resection and adjuvant chemotherapy, has been shown to result in excellent control of local disease and is, therefore, the standard treatment for advanced rectal cancer. However, the prognosis remains largely unsatisfactory due to a high rate of distant relapse, which is the most common cause of death . AccordiOne strategy to improve the side effects of the previous plan is to use induction chemotherapy before neoadjuvant chemoradiotherapy. Induction chemotherapy is associated with a higher tolerance by patients, and it is possible to prescribe a full dose of chemotherapy in this method. Other benefits of this method include shrinkage of locally advanced tumors, which facilitates surgery and early treatment of micrometastases. However, the disadvantages of this method include delays in surgery and lower utility of subsequent radiotherapy . Today, infusional oxaliplatin and FU/leucovorin (LV) are known as standard treatments for advanced CRC , 9. It hTherefore, the aim of this study was to investigate the therapeutic implications of induction chemotherapy followed by neoadjuvant chemoradiotherapy and surgery in locally advanced rectal cancer. We also used oxaliplatin and capecitabine during induction chemotherapy, as well as neoadjuvant chemoradio-therapy, and evaluated its effects and side effects.This study was performed as pilot phase 2, in which 30 new patients with locally advanced rectal cancer were enrolled; who were referred to Imam Hossein hospital in Tehran from 2018 to 2019. Inclusion criteria were age between 18 to 65 years, presence of rectal adenocarcinoma up to a maximum distance of 15 cm from the anal verge, presence of T3 or T4 in terms of TNM, or involvement of lymph nodes (according to the American Joint Committee on Cancer (AJCC) TNM staging system, eighth edition), performance status <2, glomerular filtration rate (GFR) >50, normal liver function , and bilirubin less than 3 times the maximum normal value. The study process was completely explained to all patients, and informed consent forms were obtained. Patients with non-adenocarcinoma tumors, T1 N0 or T2 N0 tumors, metastases, previous history of malignancy, history of chemotherapy or pelvic radiotherapy, pregnancy and lactation, recurrent rectal cancer, comorbidity , and any prohibition to perform chemotherapy or radiotherapy were removed from the study. Routine tests , and carcinoembryonic antigen) were done before starting treatment. 2 every 3 weeks and capecitabine 1000 mg/m2/bid for 14 days every 3 weeks for 2 courses) and then neoadjuvant chemoradiotherapy . Endoscopic ultrasonography (EUS) or magnetic resonance imaging (MRI) of the abdomen and pelvis with rectal protocol for tumor staging, computed tomography (CT) scans of the thorax, abdomen, and pelvis with and without intravenous (IV)/oral contrast, and endoscopic examination of the entire colon were performed; all patients’ information was recorded. Three patients were staged by an experienced gastroenterologist with EUS and others with MRI. Patients were first treated with induction chemotherapy . Complications of treatment were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5 criteria, and dose adjustment was performed based on the British Columbia (BC) Cancer Protocol.Statistical Analysis Data analysis was performed using SPSS 25 . For quantitative variables, mean and SD, and for qualitative variables, absolute and relative frequency were used to present data. Comparison of categorical variables between the study groups was made using the chi-square test or the Fisher’s exact test. P-value<0.05 was considered statistically significant.Initially, 30 patients were selected for the study. After chemoradiotherapy for all patients, thoracic, abdominal, and pelvic CT scans were performed with and without contrast to assess metastasis. One patient was excluded from the study due to the presence of pulmonary metastasis after induction chemotherapy. The mean age and mean body mass index (BMI) of the studied patients were 56±9.78 and 25.3±3.1, respectively. No progressive disease was reported in any of the patients. R1 resection was performed in 1 patient, and R0 resection was performed in others. In terms of tumor distance from the anal verge, lower rectal tumors are located approximately 3-6 cm from the anal verge. Mid rectal tumors are placed from 5-6 cm to 8-10 cm from the anal verge, and upper rectal tumors are placed from 8-10 cm to 12-15 cm from the anal verge. Mid-lower tumors are located in both middle and lower spaces of the rectum, and mid-upper tumors are seen in both middle and upper areas of the rectum. The rate of lower, mid-lower, middle, mid-upper, and upper tumors were 17.2% (6 patients), 13.8% (4 patients), 10.3% (3 patients), 48.3% (14 patients), 10.3% (3 patients) respectively. P=0.041).Also, in 23 patients, anal sphincter preservation was possible, which patients with lower and mid-lower tumors had the lowest sphincter preservation rate. In this study, 9 patients had lower and mid-lower tumors, and in 4 patients (44.4%), sphincter preservation was performed. It was also found that there was a significant relationship between the distance from the anal verge and sphincter preservation in patients  had side effects from induction chemotherapy, including hematologic (2 patients), diarrhea (1 patient), and neuropathic (1 patient) toxicities. Patients were re-examined for complications after completing the course of chemoradiotherapy. CRC is the third most common malignancy in men and the second most common malignancy in women, accounting for 8% of cancer-related death worldwide . The sprToday, living standards have risen dramatically across the country; over the past 3 decades, rapid socioeconomic developments have led to a change in lifestyle and a tendency to be sedentary, high-fat and high-protein diets, and reduced consumption of cereals and fiber , 29. ProDespite the growing tendency to use full neoadjuvant therapy for patients with advanced local rectal cancer, the optimal neoadjuvant treatment for patients remains controversial , 32. TheThis study was performed to investigate the therapeutic implications of induction chemotherapy and then neoadjuvant chemoradiotherapy and surgery in locally advanced rectal cancer. Finally, adjuvant chemotherapy was given to all patients. Of the 30 patients, 1 patient was excluded from the study process after induction chemotherapy due to pulmonary metastasis. Also, 2 patients did not complete the adjuvant treatment, one of which was due to the patient's failure to complete treatment, and the other was due to a grade 4 hematologic complication. P=0.04) and increased chemotherapy compliance (P<0.001) but has no effect on pCR  patients before chemoradiotherapy and surgery reduced grade 3 and 4 toxicity , which the most common complication was diarrhea (16). The results of this study are similar to our study. Five patients developed grade 3 or higher complications after induction chemotherapy, and the most common complication was hematological complications, seen in 2 patients. Fernandez et al. studied the effect of chemotherapy with capecitabine/oxaliplatin before chemoradiotherapy in rectal cancer. In this study, performed on 77 patients, a pCR level of 24% was reported. Also, the rate of complications of grade 3 or higher degrees was 11%. These results were similar to our results . Also, in patients with lower and mid-lower tumors, there was the lowest rate of sphincter preservation. Chau  results . In addiotherapy . These rA study of patients with rectal cancer found that keeping the sphincter significantly correlated with multiple factors such as age, BMI, total infiltrated circumference, tumor distance from the anal verge, and depth of invasion. Therefore, it was found that a 5-cm tumor distance from the anal verge is the boundary for the selection of sphincter-preserving resection surgery treatment. Other studies have also shown that tumor distance from the anal verge is the most important factor for considering sphincter-preserving surgery , 41. Our study has some limitations. Due to the selection of samples from a referral medical center, the generalizability of the results is reduced. If the results are evaluated on the basis of high-risk factors, such as mesorectal fascia involvement, T4 or N2, or lower rectal tumors, it is possible to achieve more accurate results in these patients. In addition, because the research was conducted as a pilot study, the number of samples examined was small. Also, designing more complex studies with a control group to better compare the addition of induction chemotherapy to neoadjuvant chemoradiotherapy with only neoadjuvant chemo-radiotherapy in locally advanced rectal tumors will provide a better understanding of this treatment. The patient follow-up to assess the impact of induction chemotherapy before chemoradiotherapy on patients’ survival may be the subject of future research. Overall, based on the results of this phase 2 pilot study, it is inferred that induction chemotherapy, followed by neoadjuvant chemoradiotherapy and surgery, is effective in locally advanced rectal cancer and has acceptable toxicity. Therefore, the use of this treatment in these patients is recommended.


A simple and efficient process for fabricating customized aspheric lenses is reported, in which a stereolithographic 3D printer combined with the meniscus equilibrium post-curing technique is employed. Two kinds of UV-curable resins, DentaClear and HEMA, were used for printing aspheric lenses in our experiments. The printed DentaClear lens featured low surface profile deviation of ~74  Aspheric lenses are increasingly applied in various imaging systems to reduce spherical aberrations, distortion, and coma, as well as to correct pupil aberrations. Using aspheric surfaces instead of traditional spherical lenses can significantly reduce the number of optics in imaging systems . The preThree-dimensional (3D) printing, referring to the technology of fabricating 3D parts layer-by-layer from computer-aided design (CAD) models, is a promising method for fabricating elements that are challenging using traditional technologies, such as vascular stents , microflIn this paper, we report a simple and effective way to manufacture customized aspheric lenses with a stereolithographic 3D printer. Commercial UV-curable resin DentaClear and built-in-house resin HEMA (2-hydroxyethyl methacrylate) were utilized to print the designed aspheric lenses. The printed lenses were post-processed by meniscus equilibrium post-curing using the same resin, respectively. The printed lenses were characterized using optical microscope, laser confocal microscope, stylus profilometry, and atomic force microscopy (AFM). Furthermore, the imaging properties of the printed lenses were evaluated with the resolution chart.Two kinds of UV-curable resins, namely DentaClear and HEMA, were utilized to form aspheric lenses. As shown in −1 Pa−3 PaThe viscosity of DentaClear was measured to be 6.08 Ten samples with a diameter of 20 mm and a thickness of 1 mm (5 DentaClear samples and 5 HEMA samples) were tested for transmission in the spectral range of 200~1500 nm . 2 is plotted in The curing thickness of the resins with an exposure time at a constant power of 32 mW/cmR represents the radius of curvature, An aspheric lens was designed and optimized using commercial optical design software to reduce image distortion and spherical aberration. The aspheric surface profile can be expressed by:R, was calculated to be −22.62, which defined an axisymmetric quadric. The aspheric coefficients, iA, which represent the deviation of the surface from the axisymmetric quadric, were calculated to be A04 = 2.25 × 10−5 and A06 = 3.13 × 10−9. Imaging resolution of the optimized lens was analyzed in terms of the point spread function (PSF). In λ is the wavelength of light and NA is the numerical aperture of the lens. Based on the above-mentioned formula, the theoretical lateral imaging resolution of the designed aspheric lens is about 1.69 The diagrammatic sketch of the optimized aspheric lens is shown in h, while the width, ib, of each corresponding layer is calculated from the layer thickness, h, and the equation of the designed lens surface . This printer has a bottom-up configuration, that is, the curing light with a wavelength of 385 nm is projected from underneath the resin tray to polymerize the resin. The building process is shown schematically in The printed lenses were immersed into the same resin from which they were composed after they were finished printing. Then, the lenses were lifted out of the resin to completely stabilize the liquid meniscus. Following that, the lenses were transferred into a UV-curing machine to solidify the meniscus. The lenses were finally cleaned in isopropanol solution with an ultrasonic cleaner so as to remove residual uncured resin. This post-curing process can be finished within 10 min. After the post-curing process, printed surfaces become much smoother.ch, in Equation (2):ch, in Equation (2) is the maximum height that the fluid can reach on an infinite vertical wall, as shown in b0. The contact angle, Theoretical simulation was performed to analyze the effectiveness of the meniscus equilibrium post-curing for surface smoothing. As described in ,16,17, tch and ch, of DentaClear and HEMA. It can be estimated that for DentaClear, ch = 1.7 mm, and b0 = 2.5 mm. For HEMA, ch = 1.2 mm, and b0 = 1.3 mm. In our experiments, the layer thickness during the stereolithography process was h = 0.01 mm. Based on the layer thickness and the equation of the designed lens surface, the width of the corresponding horizontal planes for each layer, ib, was computed, ranging from 0.0247 to around 0.3428 mm, where for both DentaClear and HEMA, h < ch and ib < b0. The boundary conditions of Equation (2) for all the layers would be:For a given liquid–solid system, both parameters  using MATLAB. The surface profiles of the smoothed lenses can be obtained by combining the solved meniscus curve of each layer. As the layer thickness, The surface properties of the printed lenses were characterized using optical microscope (OM), laser confocal scanning microscopy (LCSM), stylus profilometry, and atomic force microscopy (AFM). The printed lenses before and after the smoothing process were photographed . The lenThe surface morphology was also observed with an optical microscope , as shown in The surfaces were also characterized with a laser confocal scanning microscope . The results of the surfaces after smoothing are presented in The surface roughness and surface form of the lenses were further measured using stylus profilometry . The stylus was a conisphere diamond tip with a 2 μm radius. Moreover, the surface roughness of the smoothed DentaClear lens was further tested using AFM . Referring to As shown in mination c. The suWe have described a process for fabricating customized aspheric lenses, in which a stereolithographic 3D printer is combined with the meniscus equilibrium post-curing technique. The effectiveness of the meniscus equilibrium post-curing for surface smoothing was theoretically analyzed, and the results show that the deviations between calculated profiles and the designed profiles range from −0.89 to around 7.48 Post-curing can smooth the printed lenses of both DentaClear and HEMA, and appeared to be more effective for DentaClear. The profile deviations for DentaClear and HEMA from the designed profile were ~74 and ~150 μm, respectively. The surface roughness was also evaluated by means of stylus profiler, AFM, and LCSM. The surface roughness of DentaClear (0.16 μm) was superior to that of HEMA (0.43 μm). The printed DentaClear lens can resolve a target of as fine as 50.80 lp/mm, i.e., res = 4.92 In summary, we have demonstrated that stereolithography 3D printing combined with the meniscus equilibrium post-curing technique is a simple and effective approach to create customized aspheric lenses. The commercial DentaClear lens is of low surface profile deviation and has satisfactory optical imaging resolution. However, the HEMA lens was of rough surface and poor optical performance. Further experiments should be carried out to optimize the resin composition. In addition, the mechanical and optical properties of the printed lens should be further improved for the wide application of 3D-printed lenses. For example, by investigating new formulations of photocurable resins, as reported by Kotz et al.  and Moor


Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers’ revenues and the availability of local fish, causing a major economic loss in Italy’s main shellfish sector. Methods: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. Results: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. Conclusion: In this regard, it is essential to develop and activate, as soon as possible, an “early warning” system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations. Protoceratium reticulatum (Clap. and J. Lachm.) Bütschli 1885, Lingulodinium polyedra (F. Stein) J.D. Dodge 1989, and Gonyaulax spinifera (Clap. and J. Lachm.) Diesing 1866 [Phytoplanktonic dinoflagellates produce polycyclic ether compounds, yessotoxins (YTXs), which accumulate in filter-feeding organisms . The maiing 1866 ,3,4. AllSince the initial discovery of YTX, many structures have been discovered and described, such as 45-hydroxyYTX, carbonYTX, 1-desulfoYTX, homoYTX, 45-hydroxyhomoYTX, and many others. More than 90 analogues have been suggested for YTXs, many of which have had their chemical structure fully identified in recent years, while others have not yet been clearly defined ,8,9,10.Patinopecten yessoensis, a cold-water species of marine bivalve, common to the northwestern Pacific [YTXs were first isolated in 1986 from scallops of the species  Pacific . Initial Pacific , in contIn 2002, the European Union, through Decision 2002/225/EC, separated the marine biotoxins of the DSP complex into its four constituent categories: okadaic acid (OA) and dinophysitoxins (DTXs), yessotoxins (YTXs), pectenotoxins (PTXs) and azaspiracids (AZAs). It also established the methods of analysis and maximum permitted limits in shellfish intended for human consumption, for the different groups of lipophilic biotoxins .Yessotoxin (YTX)  is a polThe mechanism of action is still currently under investigation but point to calcium homeostasis .Azaspiracids (AZA) are six-membered cyclic amine polyether toxins, featured by a unique tri-spiro-assembly and a carboxylic acid group, which gave rise to the name Azaspiracids  Scheme . RegardiEuropean legislation established until 2013, with Regulation 853/EC/2004, a maximum tolerable limit of 1 mg of yessotoxin equivalent/kg; this regulation was amended in 2013 and the tolerance limit was increased to 3.75 mg of yessotoxin equivalent/kg, with Regulation 786/EC/2013 currently in force.Shellfish become contaminated due to their intense filtering activity. YTXs accumulate most in the hepatopancreas of molluscs, while limited absorption was found in the gastrointestinal tract. In humans, there have been no reports of adverse effects unequivocally attributable to YTXs, even when they are present in molluscs in high concentrations, but in mice, when administered intraperitoneally, they are toxic ,19. In pHaliotis midae) was reported in 2017 during a flowering of Lingulodinium polyedra and Gonyaulax spinifera, both of which produce YTXs [In South Africa, the mortality of millions of abalone . This is in order to highlight the trend of YTXs presence from 2012 to 2020 following the replacement of the biological test (MBA) with the chemical method liquid chromatography with tandem mass spectrometry (LC/MS-MS), and to verify, thus, a new trend of the same.At the same time, the present work also aims to highlight the prolongation over time of the presence of potentially toxic algae .Lingulodinium polyedra was the most abundant and most consistently present, Gonyaulax spinifera was the second most frequently reported species and, finally, Protoceratium reticulatum was the least frequently found species  and these temperatures have been maintained at these levels for longer and longer periods, probably due to global warming. As demonstrated by Guerrini et al. , the proP. reticulatum blooms in northern Japan was reported [Chemical and physical characteristics of the Northwestern Adriatic seawaters are highly variable as the basin has a low depth and receives considerable freshwater discharge, mainly from the Po river, that significantly affects both water circulation and nutrient balance. Many studies documented the occurrence of dinoflagellate blooms after freshwater inputs following either river discharge or heavy rain falls ,29; recereported .The health surveillance plans for molluscs, as provided for in Regulation 853/EC/2004 and subsequent amendments and integrations in the last regulation EU 2019/627, require the sampling of mussels on a weekly basis. The limit of yessotoxin equivalent/kg is currently set at 3.75 mg, which came into force in September 2013, while previously it was 1 mg/kg.This means that the maximum toxin value was exceeded from October 2012 to January 2013, with the last exceedance in April. Even though the value set for the yessotoxin was increased, the toxicological criticality was nevertheless repeated following the accumulation of this biotoxin in mussels, with concentrations above the new limit from January to April 2015. The non-conformities recorded were managed by issuing separate bans on mussel harvesting due to yessotoxin.It is very interesting to observe the course of algal proliferation over the years, although this has not always led to the production of biotoxins. We can ascertain a trend of seasonal growth of microalgae. They appear every year, with varying degrees of intensity, from May onwards and continue to appear until October, a sign that their multiplication is influenced by rising water temperatures.Mussel harvesting stoppages caused by algae negatively affect farmers’ revenues and the availability of local fish, leading to a major economic loss in Italy’s leading shellfish sector.High levels of yessotoxins are a major danger to consumers. The insidiousness lies in the mechanism of action of this toxin, as it is still unknown and does not allow us to have reliable information on the symptoms and problems that it develops in humans.The data, which are recent, are intended to draw attention to the issue of harmful algal blooms (HABs), which are now constant in our seas.YTXs first appeared on the northwest coast of the Adriatic Sea in 1995. In 2004, YTXs were detected for the first time in mussels from the northeastern Adriatic Sea. More recently, YTX has been confirmed as the most common biotoxin in the eastern-mid Adriatic Sea. Recurrence of yessotoxin (YTX) has changed the current view that DSP toxins are the main cause of shellfish toxicity in the eastern Adriatic Sea . Since tIn this regard, it is essential to develop and activate an “early warning” system as soon as possible, which would allow better control of the production areas of live bivalve molluscs, therefore allowing optimal management of the plants in these critical situations.Finally, regarding the production and marketing of products of animal origin, including live bivalve molluscs for human consumption, it is essential that producers comply with the indications contained in Directive 41/EC/2004, implemented by Legislative Decree No 193 of 6 November 2007.The last two regulations to consider are number 1169/EC/2011, concerning the provision of food information to consumers (label), and Regulation 1369/EC/2017, establishing a framework for energy labelling and providing us with the expiry date, which are very important to consult when purchasing a product.Given the topics covered in this ever-developing thesis and the increasing trend of algal blooms, simultaneous with the resulting economic issues, new insights into both algal blooms and the toxic capabilities of yessotoxins will certainly be needed in the future.The study area is located in the northwestern Adriatic Sea, between the mouth of the River Po and the mouth of the River Rhine. Data from 2011 show that almost 90,000 tonnes of shellfish are produced in this area every year .Mytilus galloprovincialis) and secondary oysters (Crassostrea gigas); (2) Lupini: the coastal marine area including seawaters between 1 and 2 nautical miles that is classified as class A area, in which natural banks of Mediterranean striped clams  are present and harvested; (3) B-Out: the narrow sea coastal area and inland waters classified as class B area; (4) B-In: class B area that includes the inner channels directly connected to the sea, together with internal waters; (5) Sacca di Goro : the class B area included between the Po river and the marine coastline. All these last three sub-areas were used to breed Manila clams (Tapes philippinarum). The detail of monitoring stations are shown in Five sub-areas of molluscs production have been envisaged : (1) LonChamelea gallina); 732 mussels ; 66 oysters (Crassostrea gigas); and 773 veracious clams (Tapes philippinarum). Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters.The Health Surveillance Plan on edible lamellibranch molluscs in Emilia-Romagna has been regularly applied since 1997, as provided for by Community legislation. The sampling frequency varies according to the mollusc species to be analysed and their filtering capacity . Depending on weather and sea conditions, mussels are sampled once a week, clams once a month, and common clams once a quarter. Until 2012, the determination of lipophilic marine biotoxins was done with the MBA, a semi-quantitative test based on the time of death of the mouse after intraperitoneal inoculation of a lipid extract of mollusc. Since March 2012, our laboratories have started to apply the chemical method in LC/MS-MS. For the present work, the results obtained in the period 2012–2020 were considered. In the nine years considered, 3359 samples were examined : 1715 maThe method adopted  for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. Toxins belonging to the YTX, AZA and PTX groups are identified and quantified by analysing a methanolic extract of the mussel homogenate, while the determination of total okadaic acid derivatives and DTXs is performed on the extract after alkaline hydrolysis. LC-MS/MS is conducted from a methanolic solution from homogenised tissue to obtain data regarding the extraction of okadaic acid, pectenotoxins, azaspiracids and yessotoxins. The extraction product was filtered and analysed by liquid chromatography combined with mass spectrometry; a fraction of this extract was subjected to alkaline hydrolysis and analysed, in order to quantify the presence of total okadaic acid derivatives.Toxin quantification was performed by calibration of the LC-MS/MS system with standard toxin solutions of known concentration. The certified reference materials used for calibration of the system for OA, DTX1, DTX2, PTX2, YTX, homo-YTX, AZA1, AZ2, and AZA3 were purchased from the National Research Council of Canada (NRC).The LC-MS/MS procedure was performed using an Agilent 1290 HPLC instrument coupled to an Agilent 6460 triple quadrupole, in gradient elution and negative electrospray ionization.Samples were stored in a refrigerator or freezer before analysis. In order to avoid toxin degradation processes, samples were analysed in the shortest time.For successful testing, the mollusc samples must be stored in a refrigerator or freezer. At the beginning of the test, the molluscs were washed thoroughly with fresh water, paying particular attention to their exterior, and then opened by incision of the adductor muscle. The extracted pulp was left to drain and then homogenised using a blade homogeniser. The dissection steps were then carried out and the adductor muscles were then cut to open the shells. The inside of the mollusc was rinsed with fresh water to remove sand and foreign material. Finally, the flesh was removed from the shell by separating the adductor muscles and the tissue connected to the hinge.Heat or anaesthetics should not be used before opening the shell. After removing the shells, the tissues were left to drain in a sieve to remove any remaining salt water.For representative sampling, at least 100 to 150 g of tissue was taken and homogenised in a blade homogeniser.g rotations per minute or higher for 10 min at a temperature of 20 °C, and the supernatant was then transferred into a 20 mL volumetric flask. The extraction of the residual tissue was repeated with a further 9 mL of methanol and homogenized for 10 min, again at a temperature of 20 °C, then transferred and combined with the first extract, made up to volume with 20 mL of methanol.For sample extraction, 2.00 g ± 0.05 g of homogenised tissue was accurately weighed into a 50 mL centrifuge tube; then, 9.0 mL of methanol was added and the sample homogenized, using a shaker, for 3 min at maximum speed. Separation was operated with centrifuge at 2000 × The extract thus obtained was analysed by LC-MS/MS to quantify the presence of toxins of the YTX, AZA, and PTX groups, while the quantification of total AO derivatives requires alkaline hydrolysis of the extract.Hydrolysis involved adding 125 µL of 2.5 M NaOH (sodium hydroxide) to an aliquot, exactly measured, of the methanolic extract, vortexing for 30 s and heating to 76 °C for 40 min. Subsequently, the mixture was cooled to room temperature, neutralised with 125µL HCl 2.5 M (hydrochloric acid) and homogenised by vortexing for 30 s. The extract was filtered through a 0.45 µm methanol-compatible filter or through a 0.2 µm syringe filter and, depending on the sensitivity of the instrument, between 5 µL and 20 µL was injected into the mass spectrometer column.3COONH4 and 0.1% CH3COOH/MeOH; 95:5, v/v) and a mobile phase B (2 mM CH3COONH4 in MeOH). Only LC-MS grade solvents and reagents were used.Reverse-phase liquid chromatography was performed using Poroshell 120 EC-C18, 2.1 × 100 mm, 2.7 µm C18 column (Agilent Technologies), and a binary pump system working at a flow rate of 0.4 mL/min with an injection volume of 10 µL. The gradient set up for the chromatographic separation used a mobile phase A . YTX concentrations were determined by a five-point calibration curve using dilutions of a certified YTX and homo YTX standard solution, NRC, Halifax, NS, Canada). Concentrations of the YTX analogues were expressed as YTX equivalent.Analyses were carried out in multi reaction monitoring (MRM) mode with negative ionization ; selecteThe water samples taken from 2012 to 2017 were carried out with the aid of a phytoplankton net sampler, equipped with a 20 µm mesh; from 2017 to the present, the hose-sampler has been used, i.e., a rigid tube was used, consisting of one metre segments, which were inserted into each other depending on the depth of the area to be sampled. In the Sacca farms, where the depth ranges from 1.5 to 2.5 m depending on the tidal excursion, 1-m-long tubes were used, while in the long-line farms, where the depth is about 14–16 m, 4-m-long tubes were used, i.e., tubes about as long as the mussel remains.Within two to three hours after sampling, the water samples arrived at the laboratory where they were immediately fixed with Lugol’s fluid. The Lugol’s iodine-fixed samples were examined by inverted light microscope . The procedure is described in the European Standard EN 15204:2006 and is based on the standard settling technique as defined by Utermöhl in 1958. It allows the estimation of the abundance and taxonomic composition of marine phytoplankton by using inverted light microscopy and sedimentation chambers.Before pouring the samples into the sedimentation chambers, it is very important to make them homogeneous by gently inverting the bottle containing the water to be examined at least 100 times . SedimenFor routine sampling as part of a toxin-producing phytoplankton monitoring programme, plankton nets with a mesh size of 20 µm were used. A vertical haul was taken from the depth of the mussels farm up to the surface. At the sampling site, from the boat, the plankton net was lowered; when the net reached the desired depth, it was raised slowly. Samples collected by plankton nets are inappropriate for quantitative analysis, but filtering a large amount of water can enable the early detection the toxic species even if they are in low concentrations.Hose samples are appropriate for quali-quantitative analyses of phytoplankton. The procedure used to collect the hose samples is well described in Mendez et al., 2016 .