Objective: Findings among studies evaluating the effect of statin use and OA development in a 2020 meta-analysis of data from 11 observational studies of statin use and osteoarthritis (OA) revealed controversial results. We aimed to determine the associations between statin use and OA-related outcomes in an updated meta-analysis.Methods: The protocol was registered with PROSPERO (CRD42020163983). A systematic literature retrieval was performed in the online databases, including PubMed, Cochrane Library, Embase, Web of Science, and Scopus, from inception to 1 June 2022, for clinical studies that compared the effects of statin users vs. nonusers on OA-related outcomes risks. Systematic reviews and meta-analyses were performed to estimate the correlations between statin use and OA-related outcomes. Tendency analysis was also used to estimate dose-response effects. The risk of bias was evaluated with the Newcastle–Ottawa scale.Results: We included 23 studies involving more than 6,000,000 participants. Statin use was associated with increased OA risk . Higher statin doses had higher OA risk . OA and related surgery risks were significantly reduced in statin users using antihypertensive drugs (AHDs). No significant differences were seen in other outcomes.Conclusion: This meta-analysis inferred that statin use might be associated with increased OA development, especially at higher doses. The present study highlights the importance of recognizing potential OA risk in the population with long-term and/or high-dose statin use, especially in older populations. In addition, AHDs are associated with lower OA risk and fewer surgeries in hypertensive statin users. Due to limitations of heterogeneity and confounders, more rigorous studies are needed to define the correlations between statin use and OA-related outcomes. Osteoarthritis (OA) is the most common degenerative joint disease, and the major cause of joint pain and disability. OA is increasingly recognized as worldwide health concern due to low quality of life and huge social and economic burden . OA pathStatins are currently the most effective drugs used as lipid-lowering agents. In clinical practice, statins are commonly prescribed in cardiovascular diseases and dyslipidemia treatments, especially in older populations with multiple geriatric and metabolic diseases. In addition, statins have been shown to function as agents of anti-inflammation, offering cartilage protection . Hence, A 2020 meta-analysis looking at the association between statin use and OA development and progression provided controversial results due to multiple limitations . MeanwhiThis study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines  and guidPopulation/patient: participants using statins with controls from the same population without statin medication prior to identification; intervention: statin use; comparison: statin users vs. nonusers on OA-related outcomes; outcome: data concerning OA-related outcomes, including statin-related OA risk; OA-related surgery; duration and dosage of statin use and OA risk; study design: clinical randomized/case-control/cohort studies. Non-clinical studies, studies with potential bias in participant populations, or studies without available data or conclusion  were excluded. This part of the study was independently performed by two investigators in duplicate and double-checked by a third investigator. Discordant judgments were addressed by open discussion with the research team and resolved by consensus.Study selection was based on the PICOS statement. Two investigators independently performed the data extraction and quality assessments. From each included study, we extracted information such as demographic data, conclusions and clinical significance. If multiple studies were analyzed with potential overlapping participants, we retained studies with the most detailed methods, the most participants and the most adjusted confounding factors. If multiple non-overlapping data were found, the data were allowed and regarded as independent.The risk of bias of included studies was assessed with the Newcastle–Ottawa scale (NOS) , which eData of statin users vs. nonusers on OA-related outcomes risks were calculated, including data concerning OA-related outcomes, including statin-related OA risk; OA-related surgery; duration and dosage of statin use and OA risk. OA diagnoses were defined by radiologic (Kellgren-Lawrence grade ≥2) or clinical evidence, and endpoint events were defined as OA-related outcome events. Propensity score-matched data were preferred for the entire analysis, then data of the largest number of confounders adjusted, followed by raw data. Ratios and eligible data were used. Hazard ratios (HRs), odds ratios (ORs) and relative ratios (RRs) in this study were used, deemed as homogenous and analyzed together.When combination or transformation of study estimation values were needed, in the situations like multiple co-existing estimations obtained from the same reference control group, the method developed by Hamling’s study was used when participant numbers at every level were available . Otherwi2 values. Pooled data had low heterogeneity if p was greater than 0.1 and I2 was less than 50%. In this case, a fixed-effects model was used; otherwise, a random-effects model was used  . Heterogwas used . StatistSensitivity analysis were performed to reduce heterogeneity and detect additional potential correlations. Subgroup analyses were performed in the overall cohorts where significant heterogeneity was detected and were based on extracted data. Egger’s linear regression and Begg’s rank correlation tests were performed to evaluate potential publication bias for outcomes with more than 10 included studies .For data deemed ineligible for meta-analyses, we listed and re-analyzed all data without statistical syntheses; however, the strengths of this evidence were considered to be inferior to those of the meta-analyses.The systematic literature search originally retrieved 1574 unique citations. A total of 23 studies (20 cohort studies and three case-control studies) met the eligibility criteria and were included in this study  Beattie. Most ofBased on the extracted data and transformed data, statistical analyses were performed in several cohorts, including OA incident risk (risk cohort), OA-related surgery risk (surgery cohort), duration and dosage of statin use and OA incident risk (duration/dosage cohort), OA progression risk (progression risk) and antihypertension drug and statin co-use and OA incident/surgery risk (AHD cohort). The cohort outcomes and evidence analysis of non-meta-analyzed studies are displayed in p = 0.045]). Similar outcomes were seen in the raw data   data and raw numbers. Statin use was detected as being associated with slightly higher OA risk in overall adjusted estimations  . After n 0.063]) .p < 0.001]) but not in adjusted data.For estimations concerning statin use and OA-related surgery, five candidate studies were selected where an OA-related surgery  was defined as the endpoint event. Statin use was linked to a higher number of OA-related surgery in raw data  and adjusted estimations . No significant outcomes were detected in either analysis, indicating that statin use was not associated with OA progression or radiologic progression , but the definition was not clear. To further estimate the continuous tendency of duration–risk correlations, Pearson’s correlation coefficients and fitting curves were calculated and visualized with the ggplot2 and ggpubr packages . Comparisons of the estimations were made every two adjacent durations, and meta-analyses of these ratios were performed. No significant outcomes were found, indicated that when other factors were consistent, statin use duration was not associated with OA risk.The links between statin use duration and OA risks were analyzed in nine studies. Significantly higher risks were only detected in cohorts of “former statin use” . This negative correlation was not observed in the >5-year cohort . However, a higher risk of OA was seen in the >5-year cohort , and meta-analyzed estimations in the same persistence of >5-year vs. >1-year was significantly increased   . No othep = 0.021]) and high doses  of statin use were significantly associated with higher OA risks. A sudden increase in ORs was seen in the high-dose subgroup compared with the other dose groups, suggesting that a dose threshold could exist beyond which higher doses are associated with significantly higher OA risk. When 20 mg/d simvastatin equivalent was set as a threshold, overall >20 mg/d vs. <20 mg/d of statin use calculated by meta-analyzing the estimations of a higher dose vs. adjacent lower one, higher statin use was associated with increased OA risk .Dose analysis was tested in five studies. Based on the provided data and statin dose equivalent conversions , two subp = 0.8, R = 0.057) (p = 0.049]).For the tendency analysis of dose-risk estimations from the low-dose to high-dose, positive trends that approached significance were detected (= 0.057) . HoweverCochran–Armitage tests for trends were performed using the DescTools package for non-meta-analyzed studies where binomial case and control numbers of every dose level were available. Two studies  indicatep = 0.020]), surgery ) and EST-2 , surgery ), but these outcomes may be only available for hypertensive statin users , and among those variables, race, lesion, follow-up periods and NOS scores accounted for 54.45%, 38.75%, 43.41% and 18.06%, respectively. Publication bias tests were only conducted in risk analysis, and bias was not found in Egger’s or Begg’s tests for any cohort.Beyond using statins as first-line medications to treat atherosclerotic cardiovascular events and dyslipidemia, the anti-inflammatory and cartilage-protective effects have been reported by numerous studies. Our meta-analysis comprehensively investigates whether statin use leads to OA-related outcomes, and suggest that statin use may be associated with OA-related outcomes, especially at higher doses. In addition, AHDs significantly decrease OA and related surgeries risks in hypertensive statin users.In the previous meta-analysis , methodoThe overall outcomes indicated that statin use may slightly increase the risk of OA, which was inconsistent with the previous meta-analysis. Statins have been reported to protect cartilage, and views are presented. First, cholesterol and fat were shown to be positively correlated with OA severity . StatinsAs statins are widely and long-termly used in older people, their potential contributions to musculoskeletal disorders deserve considerable attention. Some studies reported that statin use was linked to musculoskeletal conditions, including arthropathies . StatinsFor statin use and OA-related outcomes, people taking higher statin doses are more likely to develop OA. High-dose statin use may increase SAMS development, and simvastatin >40 mg/d equivalents was associated with higher osteoporosis and diabetes risks . The staThis study was performed following recommendations for rigorous meta-analyses. A major strength was the inclusion of large sample size comprising multiple population-based studies performed under rigorous research conditions or from databases based on credible records. This process provided an advantage by eliminating biases. Therefore, we feel that our meta-analysis outcomes are robust and valuable for further investigations, especially in patients with MetS. In our study, multiple statin use analyses were performed using major parameters, such as duration, doses, and multiple OA-related outcomes. We also used tendency analyses for dynamic estimations.Although several challenges were encountered and resolved, there were also several significant limitations. First, as a limitation of drug-related meta-analysis of multiple retrospective large-sample studies, it is premature to arrive at an absolute causality. Heterogeneous baseline conditions, and confounding factors and methodologies were present, and heterogeneity could not be completely eliminated. Therefore, the conclusion was limited and challenged by inconsistent quality and multiple confounders in the included studies, let alone the lack of a rigorous causal relationship. Second, some important definitions of statin use were lacking or controversial. For example, statin prescriptions did not necessarily qualify as use or even regular daily use, and no consistent definition of statin use was present. However, as OA was a long-term progressive degenerative disease, logically and theoretically, it may be inferred that OA progress, incident and surgery risk are significantly elevated with long-duration and/or high-dose use when statins were deemed associated with OA. Multi-dimensional analysis was only performed in one included study that used total statin dosage taken within a certain duration . Statin In this meta-analysis based on multiple population-based studies, the statistical outcomes inferred that statin use might be associated with increased OA development risk, especially at higher doses. These findings do not support the argument that statins alleviate OA risks and highlighted the importance of recognizing potential OA risk in the population with long-term and/or high-dose statin use, especially in an older population. In addition, AHDs reduced OA incident risk and OA-related surgeries in hypertensive statin users. However, because the methodologies and parameters of the included studies were various and heterogenous, more rigorous, multi-dimension studies with greater numbers of participants are needed to confirm our conclusions.


Metacognitive training (MCT) is an effective treatment for psychosis. Longitudinal trajectories of treatment response are unknown but could point to strategies to maximize treatment efficacy during the first episodes. This work aims to explore the possible benefit of using latent class mixed models (LCMMs) to understand how treatment response differs between metacognitive training and psychoeducation. We conducted LCMMs in 28 patients that received MCT and 34 patients that received psychoeducation. We found that MCT is effective in improving cognitive insight in all patients but that these effects wane at follow-up. In contrast, psychoeducation does not improve cognitive insight, and may increase self-certainty in a group of patients. These results suggest that LCMMs are valuable tools that can aid in treatment prescription and in predicting response to specific treatments. People that experience a first episode of psychosis (FEP) have highly variable outcomes, which range from sustained remission to treatment resistance from onset . DespiteCognitive behavioral models of psychosis have fostered the development of psychological interventions that target the cognitive biases involved in the genesis and maintenance of psychosis . These hMetacognitive training (MCT)  has emerThese broad beneficial effects may be rooted in that MCT intervenes over most of the cognitive biases and cognitive constructs that have established evidence of their importance in the genesis of psychosis, its maintenance, and outcomes. MCT includes sessions that work on domains of social cognition such as facial emotion recognition and theory of mind, both of which are strong predictors of outcome . LikewisA consistent finding in the literature is that MCT improves cognitive insight by increasing self-reflectivity and reducing self-certainty , the twoAlthough meta-analytic findings have supported the efficacy of MCT in psychosis ,24, thisUnsupervised learning methods such as latent class mixed models  are a useful approach for studying longitudinal trajectories of latent variables. LCMMs have the advantage of being able to capture inter-individual differences in intra-individual change over time while preserving the heterogeneity of the population . AnotherPrevious studies have used LCMMs to understand trajectories of illness or outcome ,32, but We sought to study the feasibility of using LCMMs to understand patterns of change in cognitive insight in individuals with FEP who have received MCT. To test whether this is a specific effect of MCT, we compared latent trajectories between patients that received MCT and a group of patients who received psychoeducation. Psychoeducation is a psychological intervention that has proven to be effective in promoting better social and functional outcomes , but accThis study aims to provide proof-of-concept evidence of using LCMMs to understand patterns of change in response to psychotherapy. We conducted a secondary analysis of data from a blind, multicentric clinical trial that has been published elsewhere . BrieflyThe assessment included the following:Sociodemographic questionnaire: Data on sociodemographic variables were collected on-site. Diagnosis and treatment were collected from the clinical history of the participants. We transformed the antipsychotic treatment to olanzapine defined daily dose (DDD) [se (DDD) .Clinical measures: The Positive and Negative Syndrome Scale (PANSS) [ (PANSS) ,38 was u (PANSS) ,40 was u (PANSS) , where hMetacognition: The Beck Cognitive Insight Scale (BCIS) [e (BCIS) ,42 was ue (BCIS)  was usedSocial cognition: The Internal, Personal and Situational Attributions Questionnaire (IPSAQ) [ (IPSAQ)  was used (IPSAQ) . The fac (IPSAQ) ,47 was u (IPSAQ) ,49 was uGlobal functioning: The Global Assessment of Functioning (GAF) [ng (GAF)  was usedThe Ethics Committee of each participating center approved this project.LCMMs were conducted using the R package lcmm  from theAt baseline, the MCT and the psychoeducation groups were compared with chi-square tests for categorical variables and Student t-tests for continuous variables.To compare trajectories, we used Mann–Whitney U tests, for which the effect size was reported with ranked biserial correlations.The final sample included 62 participants with complete data on self-reflectivity and self-certainty at the three points of assessment.p = 0.006). At baseline, the two groups only differed in that the MCT group had higher baseline global functioning than the psychoeducation group (t(60) = −2.857, We found two trajectories for self-reflectivity and two trajectories for self-certainty. The four trajectories are summarized in We found a trajectory (in red), henceforth “High SR” (n = 20), that grouped patients with high baseline self-reflectivity. The “High SR” trajectory included patients whose self-reflectivity was stable during the baseline and post-treatment assessments but declined at follow-up. The second trajectory (in blue), named “Improving SR” (n = 8), grouped patients with low baseline self-reflectivity. The “Improving SR” trajectory presented an improvement in self-reflectivity at follow-up, which declined to a level below baseline at the 6-month follow-up. p = 0.002) and general  symptoms, global functioning , and self-esteem . Patients in the “High SR” trajectory presented better scores in all the variables, indicating a better clinical state at the moment of the assessment.At baseline, the two self-reflectivity trajectories differed in negative , general symptoms , global functioning , and self-esteem . However, at this point of assessment, the “Improving SR” trajectory had significantly worse clinical insight .At post-test, the two trajectories still differed in negative symptoms  and general symptoms  and clinical insight .Finally, at the 6-month follow-up, the two trajectories only differed in negative  trajectory grouped patients with moderate levels of self-certainty at baseline. At post-test, the scores in self-certainty declined, but they increased again to baseline levels at the 6-month follow-up.The second trajectory , henceforth “Responsive SC”, comprised patients with very high self-certainty at baseline. The scores in self-certainty presented a steep decline at post-test but increased again at follow-up. However, at follow-up, the scores in self-reflectivity did not reach baseline levels.The two trajectories of self-certainty in the MCT group are depicted in p = 0.015). The difference in positive symptoms was maintained at follow-up , but patients in the “Initial Decline” trajectory also displayed worse general symptoms  at this point of assessment.At baseline, the two trajectories of self-certainty did not differ in any variable. However, at post-test, the “Responsive SC” trajectory had worse positive symptoms , henceforth “Low-SR” (n = 18), included patients that had a low baseline level of self-reflectivity. In this trajectory, self-reflectivity consistently declined over time. The second trajectory (in blue), henceforth “Worsening-SR” (n = 16), comprised patients exhibiting self-reflectivity that was high at baseline but declined steeply over time.p = 0.04). Participants in the “Low-SR” trajectory had a bigger proportion of patients diagnosed with schizophrenia as opposed to other diagnoses in the spectrum of psychosis.At baseline, the two trajectories of self-reflectivity differed in diagnosis . However, this difference was not maintained at follow-up. At the follow-up point of assessment, the two trajectories of self-reflectivity in the psychoeducation group did not differ in any variable.At post-test, participants in the “Low-SR” trajectory reported more personalizing bias , named “Low and Stable SC” (n = 23), included patients with low baseline self-certainty, which remained stable throughout the three assessments. The second trajectory (in blue), named “Worsening SC” (n = 11), included patients with high baseline self-certainty, which increased significantly after intervention and then decreased to baseline levels at follow-up. The graphical representation of these trajectories can be found in p = 0.035), positive symptoms , clinical insight , the 60–40 condition of the beads task , and facial emotion recognition .At baseline, the two trajectories of self-certainty differed in academic background  and negative  symptoms, worse global functioning , worse clinical insight , and less draws to decision in the three conditions of the beads task .At post-test, the Worsening SC trajectory presented more positive  and negative symptoms , clinical insight , and theory of mind .However, at follow up, the two trajectories only differed in positive , and worse facial emotion recognition than their counterparts. These differences were not apparent in the self-certainty trajectories of the MCT group. Moreover, at post-test, patients in the Worsening SC group presented fewer draws to decision in the three conditions of the beads task, which suggests a more pronounced tendency to present the jumping-to-conclusions bias.Furthermore, both self-certainty and the jumping-to-conclusions bias are strongly related to delusions ,23, and Although the scope of this study precludes us from drawing conclusions on the possible mechanisms of worsening self-certainty in response to psychoeducation, our interpretation is that there may be an interaction between unawareness of the disease, positive symptoms, and poor facial emotion recognition. These patients also had poor clinical insight and thus may interpret information on the disease as threatening and react by jumping to the conclusion that their thoughts and experiences are certain. These results suggest that patients that have social cognitive and metacognitive difficulties may benefit from starting psychological treatment with a normalizing approach that reduces data-gathering biases and improves insight, such as MCT.The findings of this study must be interpreted considering several limitations: First, the sample size in the two groups was small, rendering the study underpowered and limiting our ability to compare longitudinal outcomes. Similarly, the sample size of each trajectory precluded us from obtaining predictors for each trajectory. Finally, our follow-up data only extended to six months after the intervention, and we could not detect sleeper effects.These limitations notwithstanding, our findings support the use of LCMMs to study cognitive biases in response to psychotherapy and highlight the heterogeneous nature of psychosis. Future studies including broader samples comparing more interventions will help detect the chances of responding to a specific intervention and detect precision treatment strategies to prescribe psychological treatment based on the individual characteristics of each person.This study was not designed to identify predictors of outcomes after MCT. Rather, the present study aimed to offer proof-of-concept evidence of the added value of using LCMMs to improve our current knowledge of psychological interventions for psychosis. However, using LCMMs with larger samples may help detect what specific trajectories have better chances of responding to an intervention and what their predictors are and may identify potential moderators.Furthermore, this approach is not only valid for people with psychosis, as most current psychological interventions for mental illness have sound theoretical foundations and strong evidence supporting their efficacy.This work supports the added value of using LCMMs to understand how specific psychological interventions exert differential effects on cognitive biases while considering the variability of the patients’ responses. Specifically, we found that MCT improves cognitive insight in all participants with first-episode psychosis regardless of their baseline level. Conversely, psychoeducation does not affect cognitive insight and may trigger adverse effects in some patients. Finally, LCMMs could be a useful approach to detect predictors of response to different psychological treatments and to develop early targeted treatment for people with psychosis.


Metabolomics is a fundamental approach to discovering novel biomarkers and their potential use for precision medicine. When applied for population screening, NMR-based metabolomics can become a powerful clinical tool in precision oncology. Urine tests can be more widely accepted due to their intrinsic non-invasiveness. Our review provides the first exhaustive evaluation of NMR metabolomics for the determination of colorectal cancer (CRC) in urine. A specific search in PubMed, Web of Science, and Scopus was performed, and 10 studies met the required criteria. There were no restrictions on the query for study type, leading to not only colorectal cancer samples versus control comparisons, but also prospective studies of surgical effects. With this review, all compounds in the included studies were merged into a database. In doing so, we identified up to 100 compounds in urine samples, and 11 were found in at least three articles. Results were analyzed in three groups: case (CRC and adenomas)/control, pre-/post-surgery, and combining both groups. When combining the case-control and the pre-/post-surgery groups, up to twelve compounds were found to be relevant. Seven down-regulated metabolites in CRC were identified, creatinine, 4-hydroxybenzoic acid, acetone, carnitine, d-glucose, hippuric acid, l-lysine, l-threonine, and pyruvic acid, and three up-regulated compounds in CRC were identified, acetic acid, phenylacetylglutamine, and urea. The pathways and enrichment analysis returned only two pathways significantly expressed: the pyruvate metabolism and the glycolysis/gluconeogenesis pathway. In both cases, only the pyruvic acid  and acetic acid  were present. Colorectal cancer (CRC) is the second leading cause of cancer death in men after lung cancer and the third leading cause of cancer death in women after breast and lung cancer . To mitihttps://urinemetabolome.ca/, accessed on 22 August 2022). Another problem with urine is the normalization process.The metabolome is the complete set of small molecules  found in a biological matrix. For this study, we focused on metabolites present in urine. Urine is obtained non-invasively, is available in larger quantities than blood, and is mostly free from interfering proteins or lipids. Moreover, the adherence to a screening program is much higher than FOBT, due to its simplicity and higher acceptance. However, urine is chemically complex as it contains the waste breakdown product of foods and beverages, environmental contaminants, drugs, endogenous waste metabolites, and bacterial byproducts . CurrentNuclear Magnetic Resonance spectroscopy (NMR) and mass spectrometry are the two most widely used analytical platforms for metabolomics. Although these technologies each have their advantages and disadvantages, they may be used to complement each other ,14. The Precision medicine, also called personalized medicine, is a prevention and treatment strategy tailored to an individual, compared to the classical approach based on the principle of “one-size-fits-all,” which does not allow more advanced treatments nowadays . TherefoIn this study, with a special focus on colorectal cancer, we reviewed the metabolomics-based biomarkers from urine samples detected with NMR. In addition, we provide a detailed list of found metabolites for all studies included. Results are analyzed based on three groups: 1—case (CRC and adenomas) vs. control; 2—pre-/post-surgery; 3—a combination of both groups. A vote-counting strategy has been followed for the three groups to determine the significant compounds. For the combining group, we performed pathways and enrichment analysis.The results were divided into five parts: (1) search results, (2) characteristics of the studies included, (3) quality assurance results of the studies, (4) meta-analysis results, and (5) pathways and enrichment analysis results.The search process is shown in For the ten studies included, we prepared comprehensive tables divided on the methodology of the study , cohort The information of each cohort is also summarized . ConsideQuality assurance of the studies included was performed, including ten variables for evaluation. The quality assurance results are shown in https://pubchem.ncbi.nlm.nih.gov/, accessed on 18 August 2022) was performed. In The total number of compounds identified in the 10 studies included was 100. Each reported compound name was translated to InChIKey with the chemical translation service . These rEleven compounds are the most repeated in the literature (three times each): acetone ,25,31; cMeta-analysis results followed a vote-counting strategy. Vote counting consists of the sum of the trends reported for compounds, assigning a value of +1 if the compound behavior is up-regulated, −1 if it is down-regulated, or 0 if it is equal to the comparison group. Any compound intended to be a CRC biomarker needs to be robust, meaning that it needs to be identified in more than one study, and these identifications need to all show the same trend.Up to forty-six compounds were found to be significantly different between CRC patients or patients with advanced adenoma compared to healthy controls. Three of the included studies did not report the compound fold-change ,28,29; tThere were forty compounds found to be significantly different between CRC patients pre-surgery and post-surgery see . Of thesWe considered the second group (pre- vs. post-surgery) an analog of the CRC vs. control, as pre-surgery means the patient has CRC, and post-surgery means that the patient is CRC-free. By doing so, there were seventy-four compounds found to be significantly different between case (CRC and advanced adenoma patients) and healthy controls, or by CRC pre-surgery and post-surgery see . Of thesp-value = 0.006) and the glycolysis/gluconeogenesis pathway . In both pathways, pyruvic acid (down-regulated) and acetic acid (up-regulated) are included.The significant compounds found in the combination analysis (case-control and pre-/post-surgery), are shown in Several confounding elements might affect the obtained results in this review. Some NMR-based metabolomics of CRC profile in urine samples sometimes consider heterogeneous groups of cases . If not properly considered, these factors represent important confounding elements masking biological results. For that reason, we grouped the results for those more homogenous groups, also aiming to increase the number of studies to be compared. Another confounding element is the gender effect . We haveRegarding the technology, low sensitivity has always been the primary limitation of NMR spectroscopy. Although significant signal enhancement using cryo-probes, higher magnetic fields, and digital signal processing has improved the NMR sensitivity, many important, low-abundance metabolites still cannot be detected with today’s NMR technology. It is widely acknowledged that there are several thousand measurable or detectable metabolites in human urine, but from those, only a few hundred metabolites (the most abundant) have been reported as being reliably detected by NMR ,37. WhilOne of the biggest efforts in this review was the merging and curation of all relevant compounds from the selected articles. Each individual compound from each article was searched for its InChIKey. To harmonize compounds names, we selected them from the PubChem ID associated with the InChIKey at PubChem. We have also included several chemical identifications , so it wFor the systematic review, one hundred compounds were identified in urine samples among individuals that participated in the studies with colorectal cancer or adenomas, but only twenty-five were reported more than once. Of those, the most abundant compounds were carboxylic acids and derivatives, comprising fifteen compounds , four organoheterocyclic compounds , two organic nitrogen compounds (carnitines and cholines), one benzenoid (benzamidas), one alkaloid and derivative, and one organic oxygen compound (ketone).The studies included were divided into three groups, and the analysis of significant compounds was conducted via vote-counting. (1) CRC and advanced adenoma vs. control, which returned two significant down-regulated compounds (creatinine and hippuric acid); (2) pre-surgery vs. post-surgery patients, which returned also two significant down-regulated compounds (carnitine and pyruvic acid); (3) a combination of groups one and two, which returned twelve significant compounds, including the four significant compounds from groups one and two, but with eight new significant compounds. From the significant compounds, nine are down-regulated  and three are up-regulated . Compounds with an asterisk indicate those found in groups one and two. For group three, we conducted a pathway and enrichment analysis, and two pathways were found to be significant: pyruvate metabolism and glycolysis and gluconeogenesis.In many metabolomics protocols of urine, a common practice is to normalize the volume of samples with the concentration of creatinine. Due to this normalization, no possible significant alteration in creatinine will be observed. In our case, only two studies reported normalizing with creatinine ,31, and A combination of both groups was performed with the premise that CRC post-surgery could be an analog of a healthy condition; however, some patients after surgery have micro metastasis not detectable at the time of surgery; therefore, strictly, a few patients would not be cancer-free patients. When combining both groups, up to 12 compounds were found relevant. From those, creatinine was the most found in the analyzed studied. The mentioned significant compounds per group are also relevant in the combined analysis , along with 4-hydroxybenzoic acid, acetone, l-threonine (down-regulated) and acetic acid, phenylacetylglutamine, and urea (up-regulated).The 4-hydroxybenzoate, creatinine, and acetate had significantly different metabolite levels among bladder cancer, prostate cancer, and renal cell carcinoma , and 4-hThe pathways and enrichment analysis returned only two pathways significantly expressed: the pyruvate metabolism and the glycolysis/gluconeogenesis pathway. In both cases, only the pyruvic acid (down-regulated) and acetic acid (up-regulated) were included. We can conclude that those two compounds have an opposing effect of enhancing cancer cell proliferation (pyruvic acid) and a chemoprotective effect (acetic acid).This review aimed to highlight relevant results obtained for colorectal cancer diagnosis using metabolomics by NMR and the possible role of this approach in the clinical practice. NMR-based metabolomics is a fast, high-throughput, robust, and reproducible technique ; thus, mThe search sentence used was TITLE-ABS-KEY ((urine OR urinary OR urinate OR urination) AND  AND  AND (human OR humans) AND (NMR or {nuclear magnetic resonance}) AND ).https://pubmed.ncbi.nlm.nih.gov/), Web of Science (WOS) (https://webofknowledge.com/), and SCOPUS (https://www.scopus.com/) on 30 July 2022.This sentence was searched in PubMed  the matrix did not fit the query (no urine); (2) the studies were conducted on animals or cell lines; (3) the study was not on colorectal cancer; (4) the study was related to food or drug outcomes. There was no restriction to the study design, race, geographical area, or certain population for the systematic review.http://cts.fiehnlab.ucdavis.edu/, accessed on 15 August 2022) was used to retrieve the chemical compound information and identifiers, while any missing information was checked at PubChem . Compounds’ chemical classes were retrieved using ClassyFire [http://classyfire.wishartlab.com/, accessed on 18 August 2022).In assyFire  . Pathways and enrichment analysis were performed with MetaboAnalyst 5.0 [https://www.metaboanalyst.ca/, accessed on 24 August 2022). Finally, the incidence vs. mortality plot by country was plotted using the open-source data visualization framework RAWGraphics [https://app.rawgraphs.io/, accessed on 24 August 2022).The vote-counting plot was produced with the Amanida R-package  (https:/lyst 5.0  (https:/Graphics  (https:/


Rheumatoid arthritis (RA) is a joint-disabling inflammatory disease associated with the pathology of synovitis. Some patients with RA are difficult to treat, using disease-modifying anti-rheumatic drugs (DMARDs). Biology and targeted synthetic DMARDs (b/tsDMARDs) are options for patients with RA. Acquired immunodeficiency syndrome (AIDS) is an infectious disease caused by the human immunodeficiency virus (HIV). Adalimumab is an anti-tumor necrosis factor therapy commonly used in patients with RA. However, there are no reports or related data on patients with RA-HIV/AIDS treated with adalimumab are available. In this report, we described the first successful case of a 60-year-old HIV-positive woman with difficult-to-treat RA treated with ADA after being screened for hepatitis virus, latent tuberculosis (LTBI), and other infections. She contracted HIV from sexual exposure while on adalimumab therapy. As the patient was resistant to first-line DMARDs, she continued adalimumab along with the initiation of highly active antiretroviral therapy (HAART). The patient was treated with adalimumab therapy for a year; her CD4+ lymphocyte count was normal, HIV-1 RNA decreased, and no new infections were triggered. The patient achieved clinical remission of RA. In conclusion, adalimumab is a safe option for patients with RA-HIV and may slow the progression of HIV infection. Furthermore, HAART has the potential to reduce joint pain and fatigue in patients with difficult-to-treat RA.Adalimumab is a safe option for patients with RA-HIV, and may slow down the progression of HIV infection. The HAART therapy has the potential to reduce joint pain and fatigue in patients with difficult-to-treat RA. Rheumatoid arthritis (RA) is a joint-disabling inflammatory disease that is associated with the pathology of synovitis, and some patients are difficult to treat, using disease-modifying anti-rheumatic drugs (DMARDs). Treatment options for RA have expanded with the advent of biological and targeted synthetic DMARDs (b/tsDMARDs) . In caseAccording to data from the United Nations Program on HIV/AIDS, approximately 37.7 million people worldwide are living with HIV in 2020, of which approximately 10.2 million were untreated, and approximately 1.5 million are newly infected. Furthermore, approximately 680,000 people died from AIDS in 2020. Approximately 7,000 new HIV infections in both adults and children are reported worldwide every day. Highly active antiretroviral therapy (HAART) has dramatically altered the development and transmission of HIV/AIDS. In contrast, the worldwide prevalence of RA is approximately 0.5–1% , tofacitinib, and etoricoxib because of the ineffectiveness of multiple DMARDs, for which the patient experienced insignificant joint pain relief. The patient experienced vomiting after MTX administration. For several reasons, the patient refused low-dose steroids to relieve pain. As this patient was resistant to first-line DMARDs, we initiated adalimumab (40 mg subcutaneously every other week)  therapy after screening the patient for hepatitis virus infection, LTBI, and other infectious conditions according to the British Society for Rheumatology DMARDs safety guidelines. Compared to DMARDs, the patient responded better to adalimumab therapy. Unfortunately, the patient had a concealed history of a sexual partner with HIV/AIDS. However, her DAS28 score decreased to 4.9 after two weeks of ADA treatment, and symptoms such as joints pain and weakness were significantly relieved. Nonetheless, after RA was in control, HIV was detected in her blood, with a quantitative HIV-1 RNA test revealing values of 1.31E+5 IU/mL, accompanied by a decreased CD4+ lymphocyte count. Considering that she had difficult-to-treat RA combined with HIV/AIDS, her swollen and painful joints were uncontrolled. In addition, the patient was ineffective against DMARDs. Steroids may have accelerated the replication of HIV-RNA and induce other infections. After discussion with the patient, adalimumab therapy was continued, and HAART was initiated. HIV-1 RNA viral replication was controlled, and the CD4+ lymphocyte count continued to increase , ankylosing spondylitis (AS), psoriasis (PSA), psoriasis (Ps), hidradenitis suppurativa (HS), uveitis and Crohn’s disease (CD). Adalimumab is usually recommended for patients who have failed to respond to other treatments. As a biologic agent, the safety of adalimumab deserves clinical attention. A systematic review and meta-analysis of randomized controlled trials  showed tMycobacterium tuberculosis and atypicalum. Therefore, patients with RA-HIV should focus more on LTBI.TNF-α is an important inflammatory pathway in the pathogenesis of RA, and its levels in HIV-infected patients are also suggestive of HIV/AIDS pathogenesis. Elevated TNF-α levels are associated with increased viral replication, a subsequent increase in HIV-RNA and decrease in CD4+ lymphocyte counts . Anti-TNPreviously, we emphasized that major infections, such as hepatitis virus and LTBI, should be screened in RA patients using DMARDs and b/tsDMARDs; however, data on RA-HIV patients are scarce, and case reports or clinical data on the application of biologic therapy in such patients are rare. As HAART is clinically used, the survival of HIV patients is prolonged, and the number of patients presenting with chronic rheumatologic disease is increasing. Treatment of HIV/AIDS patients with comorbid immune diseases is challenging. To our knowledge, this is the first case report of RA-HIV treated with adalimumab. During the one-year follow-up observation, continued use of anti-TNF therapy did not increase HIV-RNA levels, CD4+ lymphocyte counts remained stable, and showed a possibility of slowing down the progression of HIV infection.Previous epidemiological studies have shown that the prevalence of autoimmune diseases (such as SS) decreases significantly after the initiation of HAART.A cohort study in Taiwan  also shoThis case study shows that adalimumab is a safe option for patients with RA-HIV, may slow the progression of HIV infection, and has the potential to reduce joint pain and fatigue in patients with difficult-to-treat RA.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.J-WC, G-SD, and W-SZ contributed to the data collection and drafting of the manuscript. M-YZ helped to revise the manuscript. TG and QX conceived the study, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Furthermore, negative correlation between LV mass and LV-GLS  and LV mass Index and LV-GLS  at rest were elucidated. This descriptive reporting provided, for the first time, a sport-specific dynamic remodeling of an entire elite national team of the Ski-Mo athlete’s left heart and elucidated differences in the dynamic deformation pattern of the left heart.Eleven world elite ski-mountaineering (Ski-Mo) athletes were evaluated for pronounced echocardiographic physiological remodeling as the primary aim of our feasibility speckle tracking study. In this context, sports-related cardiac remodeling was analyzed by performing two-dimensional echocardiography, including speckle tracking analysis of the left atrium (LA), right ventricle (RV) and left ventricular (LV) global longitudinal strain (LV-GLS) at rest and post-peak performance. The feasibility echocardiographic speckle tracking analysis was performed on eleven elite Ski-Mo athletes, which were obtained in 2022 during the annual medical examination. The obtained data of the professional Ski-Mo athletes  were compared for different echocardiographic parameters at rest and post-exercise. Significant differences were found for LV-GLS mean ( Ski mountaineering (Ski-Mo) has been accepted as a new Olympic sport for the 2026 Milan-Cortina Olympics . Ski-Mo In comparison to other winter endurance sports, e.g., Nordic Cross Country skiing or biathlon, Ski-Mo involves long uphill and downhill passages that have highly varying muscular demands. This leads to significantly different athletic features in comparison to other professional winter sports athletes . Ski-Mo The term athlete’s heart describes adaptions, namely physiological, functional and electro-physiological, caused by variable physiological sport-specific demands . A conveEspecially the analyzed innovative echocardiographic data on pre- and post-exercise conditions with particular attention to the application of speckle tracking as dynamic functional assessment of cardiac remodeling might provide further insights into the characterization of biventricular and LA function in athletes and the impact of sport-specific cardiocirculatory functional adaption in these athletes . The scientific evidence on the physiological aspects of competitive Ski-Mo athletes is still sparse. However, so far sport specific cardiopulmonary remodeling due to structural and functional adaption of the athlete’s heart has already been proven ,21,22,23The local ethics committee of the University of Bayreuth approved the study protocol (O1305/1-GB). The study was conducted in conformity with the declaration of Helsinki and Good Clinical Practice . Before n = 11; male n = 8, female n = 3) were compared for different echocardiographic parameters at rest and post-exercise. All participating athletes did not have any medical history, and their sports-related history revealed no potential risk factors for sudden cardiac death in all athletes and within their families. Eleven young elite Ski-Mo professionals, all active members of the German National Team, participating in World championships and the World Cup season, were examined and evaluated in the preseason preparation time in summer during the season 2022. No participating athlete had to be excluded from the study due to post-COVID-19 infection syndromes. All participants were evaluated for anthropometric data, 12 lead electrocardiogram (ECG) and two-dimensional transthoracic echocardiography, including strain analysis at rest and post-exercise. The obtained data of the athletes , following a standard protocol, was performed . In addiFurthermore, speckle tracking analysis of the athlete’s heart was recorded at rest as well as three minutes post maximal treadmill cardiopulmonary exercise testing (CPET) as post-exercise assessment, focusing on LV, RV and LA. In this context, we obtained the LV-GLS pattern, RV free wall longitudinal deformation (RV FW long.Def.), RV four chamber longitudinal deformation (RV 4C long.Def.) as well as phasic LA strain analysis by two-dimensional strain analysis in the apical views. The detailed phasic LA strain analysis was performed according to the recent European Association of Cardiovascular Imaging (EACVI) recommendations, including LA reservoir strain (LASr), LA conduit strain (LAScd) and LA contraction strain (LASct) assessment . The LA Each of the participating Ski-Mo athletes was evaluated for the prevalence of left and right heart valve regurgitation as part of the standard echocardiographic assessment ,30,36.t-tests, with statistical significance being accepted at p ≤ 0.05. All data are presented as mean ± SD. Pearson correlations were conducted for anatomical echocardiographic parameters as independent parameter with GLS, RV deformation pattern and LA reservoir, LA conduit and LA contractile at resting conditions as well as post-exercise in our participating elite Ski-Mo athletes. Due to the relatively small number of participating elite winter sports athletes, who were enrolled as the uniqueness of this reporting, we are not able to draw reliable conclusions by uni- and multivariate regression analyses, but we might point out interesting trends in the cohort of enrolled world elite winter sports professionals. Data were analyzed with Graph Pad Prism 8.2.1(279) . All data were tested for normal distribution via Shapiro–Wilk test. Data were tested for differences with paired The baseline characteristics and anthropometric data of the participating male and female Ski-Mo athletes are presented in In the two-dimensional echocardiographic assessment, all participating Ski-Mo athletes showed a low–normal to normal systolic LV-EF at rest estimated by the biplane Simpson method and did not show any relevant pathological regurgitation of the right and left heart valves. Only mild regurgitation at the tricuspid and mitral valves was revealed. There was no relevant systolic pulmonary artery pressure evaluated by tricuspid peak systolic velocity. The obtained LA and LV assessment did not differ in between the participating athletes. The baseline echocardiographic characteristics are presented in In comparison with data from sedentary control measurements presented by the German Society of Cardiology (DGK)  and withp = 0.0036), as presented in In the speckle tracking analysis, normal LV-GLS values at rest (−21.55 ± 3.44%) and slightly reduced values for LV-GLS post-exercise (−17.25 ± 3.39%) were elucidated. The difference between rest and post-exercise assessment was significant (p = 0.48) nor for the RV apical four-chamber longitudinal deformation  at rest and post-exercise.No significant differences were found for the RV free wall longitudinal deformation  nor the LASct analysis at rest compared to post-exercise strain pattern  revealed significant differences. With respect to standard echocardiographic parameters, LA and LV geometric properties did not show significant interindividual differences. The evaluation of functional LA remodeling by the average phasic LA strain (LAS) during all three phases of the atrial cycle revealed significant differences in the comparison of resting and post-exercise conditions. In this context, across our participating athletes neither the LASr analysis at rest compared to post-exercise strain pattern  and LV-GLS and LV mass Index  at rest could be proven, as presented in No significant correlations between LAVI and the athlete’s heart strain pattern could be revealed, whereby a negative correlation between LV mass and LV-GLS (To the best of our knowledge, this is the first feasibility study to prove functional LA response due to exercise conditions in elite Ski-Mo athletes. These peculiarities provide novel insights into the assessment of Ski-mountaineers’ heart-to-exercise conditions at altitude and improve our understanding of physiological sport-specific demands in these winter sports professionals. Our previous research has shown that the heart of Ski-Mo athletes has adapted to physical exercise by individual sport-specific remodeling due to morphological and functional assessment ,7,8. OurCardiac and physiological adaptions due to exercise in elite athletes, mainly endurance athletes, represent a hot topic, and studies addressing this topic already exist ,13,17,37RV longitudinal strain seems to provide an effective tool to assess sport-associated individual athletes’ adaption or alterations of the RV by exercise-related RV overload, even at an early subclinical stage ,53,54,55Previous research demonstrated the importance of LA remodeling as a promising tool for the volumetric and functional characterization of the LA in athletes  as well Sport-specific cardiac remodeling in extreme levels of endurance sports, such as Ski-Mo, is a previously known phenomenon displaying a continuous process with a broad spectrum of adaption during an athlete’s career ,74,75,76Our feasibility trial aims to elucidate sport-specific adaptions of elite Ski-Mo athletes, who compete as very endurant athletes with enhanced anaerobic capacity at altitude and under environmental influences. However, our study is not without limitations. Firstly, our measurements were acquired in the preseason preparation time in summer with respect to a certain deviation in individual training schedules. Secondly, the slightly anthropometric variability in the cohort of Ski-Mo athletes with a mixture of young and experienced athletes entails an intra-cohort variability and contributes to a certain standard deviation in our cardiac assessment, such as the LV Mass index. Furthermore, no comparable winter sports athletes with different ethnic backgrounds except Caucasian athletes were analyzed. Additionally, focusing on speckle tracking deformation pattern pre- and post-exercise, no complete echocardiographic examination and no calculation of average septal and lateral E’ data were obtained according to the current and latest recommendations ,34. DuriThis report provides, for the first time, new evidence of sport-specific dynamic remodeling of the Ski-Mo athlete’s heart and elucidates differences at resting and post-exercise conditions in the strain pattern of the left heart against the sport-specific altitude training background of Ski-Mo athletes.In conclusion, strain imaging is technically feasible in world elite Ski-Mo athletes and might contribute to a better understanding of sport-specific remodeling and probably enhanced physiological remodeling in these highly trained altitude athletes. Further data are warranted to characterize the sport-specific dynamic atrial and ventricular cardiac remodeling, and our obtained findings might pave the road to future studies with long-term follow-up and a greater number of athletes to verify and strengthen the scientific evidence base.


In this review, we discuss the capabilities and applications of droplet flow cytometry (DFC). This unique technique uses standard commercially available flow cytometry instruments to characterise or select individual microdroplets containing single cells of interest. We explore current challenges associated with DFC and present our visions for future development.The interrogation of single cells has revolutionised biology and medicine by providing crucial unparalleled insights into cell-to-cell heterogeneity. Flow cytometry (including fluorescence-activated cell sorting) is one of the most versatile and high-throughput approaches for single-cell analysis by detecting multiple fluorescence parameters of individual cells in aqueous suspension as they flow past through a focus of excitation lasers. However, this approach relies on the expression of cell surface and intracellular biomarkers, which inevitably lacks spatial and temporal phenotypes and activities of cells, such as secreted proteins, extracellular metabolite production, and proliferation. Droplet microfluidics has recently emerged as a powerful tool for the encapsulation and manipulation of thousands to millions of individual cells within pico-litre microdroplets. Integrating flow cytometry with microdroplet architectures surrounded by aqueous solutions , for high-throughput single-cell analysis. By adding cell sorting functionality, fluorescence-activated cell sorting 7,8 can selectively isolate individual cells of particular interest from heterogeneous populations into separate containers , allowing subsequent experiments on the selected cells. Single-cell analysis by FC (including FACS) has enabled significant advances in life and biomedical sciences, clinical diagnosis and drug development.9–11Single-cell analysis plays an essential role in revealing the differences of individual cells in structure and function.i.e., secretomes) could represent putative tumour biomarkers or therapeutic targets.12 Metabolites produced by industrial microorganisms have great values in nutrition, sustainable agriculture and healthcare sectors;13 real-time cell proliferation assays are essential in drug discovery and clinical evaluation.14,15 Therefore, there is a need to develop strategies that enable FC to make comprehensive measurements of cells, including extracellular, spatial and temporal biomarkers of biological, industrial and clinical significance.However, FC-based single-cell analysis is exclusively limited to readouts from fluorescent molecules tagged either within the cells or on the cell surface. The attributes of individual cells are directly quantified and assessed by the light emitted by the fluorophore, which indicates the number of antibodies present within or on the cell. Unfortunately, this largely excludes important extracellular biomarkers and cell phenotypes and activities that are spatially and temporally important, such as secreted proteins, extracellular metabolite production, and proliferation. For example, the proteins secreted by cancer cells  droplets, two main architectures of droplets suspended in aqueous solutions are compatible with FC: water-in-oil-in-water (W/O/W)18 double emulsion (DE) droplets and hydrogel droplets.19 The pairing of FC and droplets allows a complete characterisation of individual cells beyond the conventional surface and intracellular biomarkers, featuring high throughput, sensitivity and dynamic range, and low cost. Moreover, it enables tandem genomic, epigenomic, or transcriptomic analyses on isolated cells,20 allowing integrative single-cell analysis techniques. This integrated approach has helped a wide range of discoveries, including directed evolution of enzymes and proteins,21–24 probing cellular heterogeneity in response to drug treatment,25,26 recognition of rare cells in microbial communities,25,27–30 determination of antibiotic resistance genes,31,32 and identification of biomarkers linked to diseases.33,34Droplet microfluidics35,36 and other variants of the technique 37,38 and printed droplet microfluidics (PDM)39) allow on-chip screening and sorting of W/O droplets based on fluorescent readouts of in-droplet assays, the high complexity of custom devices and instruments involved limit their widespread adoptions.35,40 Moreover, they can only simultaneously measure 1 or 2 fluorescent parameters and requires relatively slow rates  for high-accuracy sorting.40 Besides fluorescence, other optical detection techniques, such as adsorption spectroscopy41 and Raman spectroscopy,42 have been applied to characterise and select droplets encapsulated with individual cells in a microfluidic chip. However, these techniques suffer from low specificity (adsorption spectroscopy) and low sensitivity (Raman spectroscopy), making them unpopular in high-throughput droplet screening workflows.Although fluorescence-activated droplet sorting (FADS)e.g., mammalian, bacterial and yeast cells) are encapsulated individually within microdroplets (either W/O/W DE droplets or hydrogel droplets), which can be screened and sorted by commercial FC machines, and further analysed at the downstream . This integrated technology, so called droplet flow cytometry (DFC), allows a variety of biochemical assays at the single-cell level, such as single-cell cultivation, molecular evolution, single-cell detection and cell-to-cell interactions, due to droplet monodispersity, cell compartmentalisation and high-throughput processing. Also, this opens avenues for broad applications in the fields of drug discovery, metabolic engineering, medical diagnosis and cell biology.The article reviews the current achievements in use of FC (including FACS) to screen and isolate microdroplets encapsulated with single cells of interest. It should be noted that we do not highlight advances in FADS and other techniques for screening and sorting of cell-laden W/O droplets on a microfluidic chip. As shown in Specifically, this review is composed of the following sections. First, Section 2 introduces two main architectures of emulsion microdroplets compatible with FC, namely W/O/W and hydrogel droplets. Next, Section 3 summarised the main capabilities of integrating emulsion microdroplets and FC techniques  for high-throughput single-cell analysis. Also, Section 4 discusses critical field applications of DFC. Furthermore, Section 5 discusses current challenges confronted by DFC with an eye towards future development. Finally, Section 6 concludes the review.2.e.g., a buffered saline solution) to deliver and focus cells one by one in a narrow uniform stream. It enables cells to be equally illuminated, detected and measured as they pass through the laser intercept or interrogation point. Since FC requires cells to be suspended in an aqueous sheath fluid, it is not compatible with conventional W/O droplets having an insulating oil surrounding the aqueous core. Fortuitously, two main formats of droplets suspended in aqueous solutions, W/O/W DE droplets and hydrogel droplets, provide alternate droplet architectures compatible with FC. Individual cells can be encapsulated, cultured, subjected to biochemical assays in droplets; the cell-laden microdroplets can be detected as individual events by the cytometers and then screened or selected. Flow cytometric analysis of single-cell laden microdroplets allows a detailed study of cellular characteristics at the single-cell level by combining high throughput and rapid analysis with a relatively low reagent consumption and a high dynamic detection range.An FC system is typically equipped with a fluidic system for cell handling, consisting of sheath fluid  or oil-in-water (O/W) single emulsions. There also can be more complex systems, such as W/O/W DE droplets, in which a W/O emulsion is dispersed in a second continuous aqueous phase.51 In the first approach, water phase and oil phase are mixed by stirring to form W/O droplets, which can then be encapsulated by another water phase via constantly adding more water under the stirring condition. However, DE droplets produced by this approach have large variations in size.52 The one-step continuous flow method is more widely used for single-cell encapsulation. DE production by this approach always requires a relatively high rate of oil phase flow to cut water phase with a low rate to generate W/O droplets, and thereafter a second aqueous flow with a much higher rate is used to cut the oil-water layer flow. Unlike the batch method, it ensures that microdroplets can be consecutively made with a uniform size.There are two main approaches to generate W/O/W DEs: the two-step batch method and the one-step continuous flow method using microfluidic devices.e.g., substrates, buffers, fluorescent dyes and antibodies), cell suspensions or a gene library, providing a suitable environment for different cellular assays.49,53,54 The oil shell is bioinert and non-adhesive to hydrophilic activities and cells. The usage of fluorinated oil  acting as a selective barrier can reduce hydrophilic mass transfer and communication between DEs.49,55,56 The aqueous external phase allows the suspension of DEs in nutrient medium for long-term cell culture and the compatibility with commercial cytometers for high-throughput assays18,49,57,58 .In the DE, the aqueous internal phase can be composed of a reaction mixture  of the same target, revealing the biological functions of massive genetic pathways.48–50The integration of FACS and W/O/W DE droplets have already found various applications, such as analysis of cell-secreted molecules and enzymatic turnover. Moreover, the isolated cells from heterogeneous populations allow downstream bioinformatics analysis  after gelation for use in FC. The hydrogel microdroplets can offer a biologically friendly environment . A microcapsule remains the activities of living cells61–65 and their genes,66–69 secreted proteins70–73 and other ingredients . Due to their biocompatibility, mechanical and chemical stability in aqueous media, sufficient permeability of porous structure and tunable polymer properties, hydrogels  have been widely used in droplet microfluidics for single-cell assays.Hydrogels are three-dimensional cross-linked hydrophilic polymer networks that are capable of swelling or de-swelling reversibly in water. By using monomer or polymer solutions as the aqueous dispersed phase and oil with a surfactant as the continuous phase, hydrogel droplets in oil phase can rapidly generate by either laboratory-customised 2D microfluidic devicese.g., agarose, alginate, and chitosan) or proteins . For example, agarose is a linear neutral polysaccharide extracted from specific red seaweed, which undergoes sol–gel transition depending on temperature.78 Alginate is an anionic polysaccharide typically obtained from brown seaweed and bacteria, which chelates with alkaline earth metal ions, such as calcium (Ca2+), barium (Ba2+) and strontium (Sr2+) to form hydrogels. Gelatin is a protein derived from collagen, gels via a cold-setting mechanism and remains gel-like upon heating up to physiological temperature. It allows the liquid form to remain during the cultivation period of cells  and cell recovery under mild conditions  without cell damage.79Natural hydrogels used for emulsion microdroplets are either polysaccharides , which translate into tunable chemical, mechanical, and degradation properties of hydrogels. Typically, the aqueous monomer solutions are mixed with initiator and cross-linker, followed by polymerisation or crosslinking via different methods, such as photoirradiation. For example, poly(ethylene glycol) (PEG) can be chemically modified to generate ultraviolet (UV)-sensitive PEG-diacrylate that allows photoinitiated polymerisation.80,81Besides, synthetic hydrogels, such as poly(ethylene glycol), poly(acrylic acid), poly, poly(acrylamide) and their derivatives, offer desirable, controllable and reproducible properties  and analysing cell-laden microdroplets as individual events by FC (including FACS), DFC has enabled new functionalities and diversified applications for single-cell analysis. This section aims to summarise and discuss four main capabilities of DFC, namely single-cell cultivation, molecular evolution, single-cell detection, and cell-to-cell interaction  accumulation of two microalgal species, Euglena gracilis (E. gracilis) and Chlamydomonas reinhardtii (C. reinhardtii), in gelatin hydrogel microdroplets using FACS  can be recovered after sorting for re-culture. In comparison to previous works, this research enables quantitative tracking of not only the growth of individual cells but also their metabolite production. The proposed technology can be easily integrated into directed cellular evolution pipelines for microbial strain improvement.Microdroplets can act as miniaturised bioreactors to facilitate cell-based biological studies. This is because they mimic the micro-scale environment where biochemical events transpire and significantly enhance the efficiency and sensitivity of high-throughput cellular studies by reducing reagent consumption and improving detection signals.FACS see . The poret al.84 encapsulated E. coli cells in agarose droplets and determined the minimum inhibitory concentration (MIC) of rifampicin using FACS. Spontaneous mutants resistant to antibiotic were isolated and further characterized by DNA sequencing, which identified β-subunit of RNA polymerase, RpoB, as the target. S. S. Terekhov et al.31 reported the culture of individual cells from a heterogeneous bacterial population in DE droplets to investigate the efficacy of an antibiotic, amicoumacin A (Ami), toward different microbiomes , mammalian cells have been successfully encapsulated and cultured in DE droplets for high-throughput screening. K. K. Brower et al.18 performed the phenotyping of individual cells from four mammalian cells lines, including mouse embryonic stem cells, human T lymphocytes, mouse macrophage cells, human embryonic kidney cells, and mouse embryonic fibroblasts having different morphologies and sizes (5–20 μm), in DEs (with an inner diameter of 35 μm). Using a live-cell labelling dye, Calcein Blue AM, as an indicator of cell-laden DEs, FACS measurement results successfully discriminated empty droplets from those containing cells, which showed good agreement with theoretical predictions by Poisson distribution. This high-throughput screening of DEs encapsulated with different types of mammalian cells opens up new avenues for various biomedical applications, such as disease diagnostics, drug discovery, and rare mutation detection.In addition to microorganisms .53,54,85 Single-cell FACS has already demonstrated its capability to select individual cells exhibiting desired properties from a heterogeneous cell population, significantly increasing the screening throughput (with sorting speed >107 events per hour).86 However, FACS for enzyme selection is limited to the cases where signalling molecules have restricted diffusion out of the cells or can be entrapped on the cell surface or antibody-conjugated microbeads. This limitation can be overcome by encapsulating single cells within emulsion microdroplets, which restrict the diffusion of products by compartmentalization, and the fluorescence intensity of the whole droplet can be detected by FC, which is also accurately correlated with the activity of encapsulated individual cells.Microbial production of industrial enzymes and other value-added bioproducts is attractive due to their advantages compared to conventional chemical syntheses, such as sustainability, reduction in environmental pollution, and cost-effectiveness.et al.54 first reported using the DE-FACS technique and achieved high-throughput screening of enzyme libraries , was allowed to translate in the cytoplasm or on the surface of the E. coli cells. After encapsulation and cultivation of individual E. coli cells in DEs, compartments containing the fluorescent products, encapsulated cell variants, and the gene encoding enzyme were sorted by FACS. The isolated PON1 variants showed 100 fold improvements in TBLase catalytic efficiency. These works demonstrated the use of DE-FACS for directed enzyme evolution by directly monitoring the actual levels of endogenous cellular enzymes (rather expression levels of a reporter protein). Moreover, it shows the capability to identify rare phenotypes and genotypes for analysing large populations at a single-cell level.A. Aharoni ries see . A varia54 β-glucosidase,23 arysulfatase50, G-type nerve agent hydrolase,24 protease,22 cutinase,87 cellulase,88 glucose oxidase,89 esterase,90,91 and polymerase,92 expressed in different microbial strains, such as S. cerevisiae, E. coli and Bacillus subtilis (B. subtilis). Moreover, hydrogel droplets with either core–shell  or single emulsion  structures have enabled the directed evolution of phosphotriesterase,93 hydrolytic enzymes94 for high-throughput cell cultivation and enzymatic analysis95 by integrating with FACS. For example, M. Fischlechner et al.93 utilised gel-shell microbeads with a polyelectrolyte shell to encapsulate an enzyme, its encoding DNA and fluorescent reaction product. FACS was able to identify active clones within the compartments at rates of higher than 107 beads per hour  within the droplet compartments.To date, DE-FACS has been widely used for the directed evolution of different types of enzymes, including thiolactonase,hour see . This woe.g., organic acids and vitamins, have also been screened and selected using DE-FACS. J. M. Wagner et al.96 successfully isolated Yarrowia lipolytica (Y. lipolytica) mutants, resulting in a 54 fold increase in Riboflavin (vitamin B2) production parent strain. Since Riboflavin is an innate fluorescent product, no extra fluorescent substrates were used. Also, the isolated mutants can produce more extracellular products and the total amount of products than those separated by single-cell FACS, as confirmed by absorbance assays and high-performance liquid chromatography (HPLC). Similarly, X. D. Zhu et al.97 selected a Bacillus coagulans (B. coagulans) mutant yielding 52% higher lactic acid production than its parent strain. A pH-sensitive fluorescent sensor was adopted to indicate the level of lactic acid production inside the DEs, allowing the selection of DEs containing high-yielding mutants based on fluorescence.Besides the directed evolution of enzymes, microorganisms exhibiting high yields of other industrially essential metabolites, DE-FACS has been widely used for directed molecular evolution by isolating single cells exhibiting a desirable phenotype. By combing with sequencing of the isolated variants, it provides valuable insights into the underlying mechanisms of molecular evolution. It is also obvious that this technology can be applied to other areas where analysing and sorting single cells in a high-throughput manner is needed, such as functional genomics and advanced protein engineering.3.3e.g., DNA, RNA and proteins) and single cells are essential for probing cellular heterogeneity and discovering the unbiased diversity of cells in biology, chemistry and biomedicine. However, due to the limited initial numbers of single cells, traditional single-cell sequencing approaches are time-consuming and low-throughput. In contrast, droplet microfluidics is a promising technique for encapsulating and processing individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent consumption. By taking advantage of FC (and FACS), ultra-high throughput and multi-parameter genetic analysis of a large heterogeneous population of cells at the single-cell level can be achieved to discover stochastic variations in a complex biological system.Sensitive and selective detection of single molecules  was reported by H. Zhang et al.28 Agarose droplets demonstrated the capability to carry RT-PCR products after amplification at the single-molecule level. As a result, single-cell transcriptome analysis revealed a clear difference in the expression level of a cancer biomarker gene  between different types of cancer cells (Kato III and MDA-MB-231 cells) at the single-cell level.The integration of agarose microdroplets and FACS allows the identification and sorting of rare pathogens using polymerase chain reaction (PCR) signals from individual cells. A notorious pathogen causing many food-borne illnesses, lets see . This te29 Polyacrylamide droplets were used to encapsulate individual microbial cells and trap their genomes after cell lysis, which were further converted into agarose pico-reactors for subsequent multiple displacement amplification (MDA) reaction. After PCR, the polyacrylamide–agarose hydrogel droplets containing genomic DNA with target genes were fluorescently labelled with SYBR Green and analysed by FC. E. coli XL1 genomes containing a tetracycline resistance gene present 0.1% of E. coli MC1061 genomes without the tetracycline resistance gene can be differentiated.The detection of a single gene of individual microbial genomes from a mixture of microbial cells was achieved using hydrogel droplets with core–shell structures.12,98 and immune cells26 at the single-cell level has been demonstrated. V. Turcanu et al.30 encapsulated lymphocytes in agarose microdroplets, which were functionalized with avidin to capture biotin-labelled cytokine-specific antibodies. Cytokines secreted by the encapsulated cells can be detected using a second fluorescent antibody specific to the cytokine. The analysis of several key cytokines, such as interleukin (IL)-4, transforming growth factor (TGF)-β, interferon-γ (IFN-γ) and IL-10 secreted by human peripheral blood mononuclear cells (PBMCs) and a rat keratinocyte cell line transfected with the human TGF-β gene was achieved. Also, polystyrene beads functionalized with specific antibodies have been used to capture cytokines secreted by immune cells entrapped in the droplet compartments. V. Chokkalingam et al.25 encapsulated single activated Jurkat Tet cells in monodisperse agarose droplets with functionalized cytokine-capture nanobeads. After incubation and phase transfer, the agarose beads were stained with fluorophore-labelled antibodies that bind to the cytokines, allowing flow cytometric detection of secreted cytokines  from single Jurkat T cells .The single-cell agarose droplet flow cytometry method has tremendous implications in detecting rare pathogenic cells, analysing single-cell protein secretion and profiling of cellular heterogeneity at the single-molecule level and enumerating rare cancer cells, and shows great potential for numerous applications , was co-cultured with a mate (E. coli), which led to the growth of both bacteria in droplets, and an antibiotic producer, Streptomyces venezuelae (S. venezuelae), which inhibited the growth of S. aureus as a killer. Since all the bacteria used in the experiment have different fluorescence reporters, killers and mates can be distinguished, and droplets encapsulating with killers can be selected. This technique was then used to identify rare S. aureus killers from human oral microbiota by selecting the droplets containing a high concentration of killers and a low concentration of S. aureus. Slow-growing oral microbiota species that inhibit the growth of S. aureus were isolated, and genera associating with inhibitory activity was revealed. Using the same platform, S. S. Terekhov et al.31 performed environmental microbiota communities profiling  to discover secondary microbial metabolites. The target pathogen, S. aureus, was co-encapsulated with individual cells from the heterogeneous community to identify rare antimicrobial strains. As a result, bacterial clones with inhibitory activity, especially ‘unculturable bacteria’ that cannot be recognized by standard bulk methods, were selected and identified.S. S. Terekhov FACS see . A commo33 where bacterial pathogens were co-cultured with recombinant host microorganisms capable of secreting biocatalytic antibiotics and/or secondary metabolites  and the negative control (with the secretion of inactive lytic hydrolase enzyme), were co-encapsulated with a target pathogen S. aureus, respectively. Following host S. cerevisiae growth and protein induction, agarose microdroplets were stained with a fluorogenic viability probe (SyTox Orange) that selectively stains killed target pathogens, and then analysed by FACS. Rare positive clones producing a lytic hydrolase specific for S. aureus can be isolated with an enrichment factor of up to 10 000 fold relative to the initial mixture.In addition to DEs, hydrogel (agarose) microdroplets have also been used to uncover yeast–bacterium interactions for antibiotic drug discovery,ites see . S. cereet al.34 utilised agarose gel droplets to co-encapsulate individual bacterial cells from a complex microbial community with Chlorella sorokiniana (C. sorokiniana), a candidate algal strain for biofuel production. Using FACS, viable bacteria species exhibiting the desired phenotype  were isolated and recovered. Researchers have also reported the use of DFC for the screening of co-cultures of mammalian cells and yeast cells. Individual yeast secretors  and mammalian reporter cells were co-encapsulated and cultivated in agarose microdroplets, which is followed by FACS to select clones secreting bioactive recombinant proteins. These studies demonstrated the feasibility of DFC for screening and isolation of protein variants produced by microbes, which would enable and accelerate the discovery of new functional biologics, such as cytokines, antibodies, and soluble receptors.Moreover, the interactions between microalgal and bacterial cells have been investigated. J. Ohan et al.105 uncovered mammalian cell interaction networks. MBA2 cell line (secreting interleukin-3 (IL-3)) and the human megakaryoblastic leukemia cell line, M07e , were co-encapsulated within agarose hydrogel droplets at different ratios. Flow cytometric analysis results showed that local paracrine secretion could modulate the viability of the factor-dependent M07e cells. Besides, IL-3 secreting MBA2 cells were co-encapsulated with umbilical cord blood (UCB) cells to determine the impact of IL-3 on a heterogeneous cell population, which revealed subpopulations that are primarily dependent on locally delivered IL-3 .Using the same technique, E. Tumarkin 106,107 Researchers have achieved in vitro co-culture of different types of mammalian cells to investigate the underlying metabolic processes and interactions.108 Unfortunately, despite the biological and biomedical significance of mammalian cellular interactions, there are still quite limited reports in literature regarding the use of the DFC in the study of mammalian cell communications. With further advances in droplet compartments and cell co-culture models, we can expect that wider communities will adopt DFC for cell-to-cell interaction analysis.Mammalian cell-to-cell interactions are crucial in cell development, tissue and organ homeostasis, and immune interactions in disease.4.i.e., W/O/W DE and hydrogel droplets), it enables the screening and isolation of microdroplets encapsulated with rare cells exhibiting properties of interest and characterization of unexpected cell-to-cell heterogeneity within the population. By pairing FC with emulsion droplets  within droplet compartments in response to a specific drug or a combination of multiple drugs. This reveals a heterogenic reaction that cannot be obtained by traditional drug screening methods (which are based on the average of the entire population). In addition, the use of emulsion droplets enables the analysis of a single cell's anti-drug response . Microdroplets are also used to analyse the drug response of a cancer cell line with different phenotypes, showing a clear distinction in drug response as a function of a specific phenotype. This ability to reveal different cell line responses to the same compound allows scientists to gain important information for new drug discovery and development.Due to the emergence and spread of drug resistance and new diseases, there is an urgent need for new drug development. An effective technique for high-throughput drug screening plays a critical role in finding a new drug against the target of particular diseases, where diverse compound libraries are tested under different conditions in parallel with the drug target.4.2i.e., secretomes) are a fundamental source of biomarkers.98 Although conventional immunoassays, in particular, enzyme-linked immunosorbent assay (ELISA)110,111 have been widely used to quantify protein biomarkers in bulk, the detectable concentrations of proteins are limited to generally above the picomolar range, making them unable to detect cancer biomarkers that exist in biological fluids at concentrations in the range of 10−12 to 10−16 M.112 Moreover, it is quite challenging to perform characterisation of immune responses in many cases, as it requires the isolation of individual immune cells. For instance, a diversity of T cells can be produced by human immune system, but only a fraction of them are capable of being recognised and distinguished from those infected and mutant cells. Therefore, DFC has been applied to detect low-abundance proteins secreted from single cells and to quantify single-cell immune signatures.113 Tumour heterogeneity can confound the detection and diagnosis of cancer patients, which has led to high-throughput single-cell analysis platforms to aid in the determination of cancer malignancy.114–117 Also, co-encapsulation of distinct cell types in microdroplets holds great potential for future cancer research by probing heterogeneous cell–cell interactions .The presence and the stage of a particular disease can be indicated by sensitive and reliable detection of specific biomarkers, bacteria and viruses. For example, the proteins secreted by cancer cells , allowing to establish the linkages of phenotypes and genotypes for directed evolution of functional proteins. This mimics the process of natural evolution in a laboratory setting, which relies on iterative cycles of genetic diversity generation and phenotypic selection to isolate evolved industrial strains with desired traits. DFC has been providing an efficient screening avenue to improve microbial strains for enhanced enzymatic activity, and improved production of value-added bioproducts .96Due to the limited knowledge of complicated cellular networks, directed evolution has played an essential role in improving the performance and functionality of industrially important microbes.4.4102 For example, co-encapsulation of two bacterial strains allows for discrimination between viable and non-viable cells, identifying the conditions that support the growth of unculturable cells and improved understanding of mechanisms underlying bacterial inhibitory activity.31 Moreover, co-culture of a microalgal strain with a pool of environmentally sourced bacterial cells allows the isolation of the ones that can induce a desired phenotype .34DFC opens new opportunities in cell biology by improving our understanding of how the behaviours of individual cells shape biological processes. Microbial cells from a complex community can be randomly encapsulated into microdroplets that can be used as miniaturized compartments and screened or sorted in parallel by flow cytometers to study how the community members interact. This high-throughput approach can rapidly resolve microbial interaction networks across different initial community states, population sizes, and environments, enabling a better understanding of the critical parameters shaping the structures and functions of microbial communities.5.As discussed above, the ability to screen and select microdroplets encapsulated with single cells by FC brings in new possibilities in various fields. Compared with traditional well plate assays, this integrated technique has impressive advantages, such as high throughput and low cost. However, there are still issues that limit DFC as a common technique for high-throughput single-cell analysis.5.1e.g., incubation, relocation and manipulation) without any cross-contamination or analyte loss.22 Unfortunately, there is a possible leakage of small molecules from the droplets into the surrounding oil and neighbouring droplets, which may be due to the hydrophobicity and solubility of fluorophores, formation, and transport micellular or vascular structures, or media pH.50 This results in the reduction of sensitivity and resolution of biomechanical assays performed in microdroplets.The first area that needs improvement is the transport and leakage of the molecules encapsulated within emulsion droplets. To ensure accurate and effective in-drop assays, it is essential to maintain all the contents within the droplet compartments during the process  or sugar additives into the inner aqueous phase,122 some fluorophores of widely used substrates are hydrophobic and suffer rapid diffusion or exchange between droplets. One interesting approach is nanoparticles that can interface with target hydrophobic molecules to prevent their leakage.123Although several approaches have been reported to increase the containment of small molecules within droplets, such as the use of fluorinated oils,55 There is still a need for further development of new fluorescence dyes and metabolic biomarkers that can be confined within the droplets and new biochemical technologies that can prevent the leakage of small molecules from droplets.Another approach that may circumvent this limitation is the chemical or enzymatic modification of the hydrophobic products into hydrophilic, charged molecules that cannot diffuse into the oil shell.5.2The second important challenge is the restricted access to droplet microfluidic technology. Currently, microfluidic systems are mainly designed, fabricated and operated by specialized engineering laboratories, and the applications in life and biomedical sciences achieved are based on the collaborations with engineering research groups. Therefore, more efforts should be put into commercialising simple and cost-effective instruments and surfactants to facilitate wider adoptions, allowing users without expertise in microfluidics to carry out the wide range of applications summarised above.e.g., DropSeq and InDrops by 10× Genomics, which enables single-molecule detection by measuring thousands of independent amplification events within a single sample. Moreover, there are many companies provide a wide catalogue of standard microfluidic chips for various applications, and help with design, prototyping and fabrication of full custom microfluidic chips that meet specific needs. It would be helpful to push some relatively basic instruments for individual applications  into the market. Also, it looks great to make the technology instruments capable of integration with the multi-well plate format, so that highly parallelized downstream procedures can be carried out with existing liquid handling robots. We foresee that the accessibility of basic microfluidic technology will be expanded to non-specialists in the broader research and clinical communities.We note that a few examples of droplet microfluidic technology have been successfully commercialized, 5.3124 Also, there are limited fluorescent dyes available to probe complex cell activities in microdroplets. Therefore, developing a label-free optical detection technology for sensitive detection and characterization of droplet bioreactors would be of great promise by expanding the available assays in droplet screening and sorting.Another challenge is that the screening and analysis of droplet bioreactors have mainly relied on the detection of fluorescent products. Although fluorescent labelling allows multiple readouts simultaneously with high sensitivity, it is often challenging to incorporate them into biochemical assays. The native target is not fluorescently active, and adding a fluorescent dye may cause cellular cytotoxicity, photobleaching, and interface with cellular metabolism.41,125 has been used for enzymatic activities in droplets, but it suffers from relatively low specificity. Although Raman spectroscopy has been applied to quantify single-cell metabolite and enzyme production in microdroplets,42,126 the relatively high level of noise and low sensitivity limit its comprehensive implementation in high-throughput assays. We note that recent advances in coherent Raman spectroscopy127–130 have enabled high-throughput  and label-free analysis of heterogeneous populations at the single-cell level, which holds great promise for high-accuracy cell classification.Different optical techniques have been used to analyse droplets beyond fluorescence detection. For example, absorbance and mass-based detection131,132 and planktonic cells.133 Combining with high-speed image-based sorting,134–139 this will eventually be used in the process of isolating individual cells exhibiting properties of interest. With further advances in sensitivity and specificity of label-free optical detection techniques, we expect that droplet encapsulation and compartmentalization will expand into a more versatile platform technology for high-throughput single-cell analysis.Moreover, imaging flow cytometry (IFC) has become an attractive tool for single-cell analysis in diverse biomedical fields, and a two-dimensional image signal has been employed for real-time classification and sorting of droplets encapsulating with cancer6.via collaborations between engineers and biologists rapidly by commercializing standard instruments and chemicals  for droplet generation and manipulation. We envision that further development of droplet systems and optical detection techniques should allow DFC to expand into a more typical and versatile platform technology for high-throughput single-cell analysis.The ability to encapsulate, cultivate, monitor and select single cells in a large number of droplet bioreactors provides new opportunities in tackling numerous problems in fundamental and applied biology and health. New toolsets and new fluorescent toolsets may deliver breakthroughs in tackling the challenges regarding transport and leakage of small molecules in droplets. The availability of droplet technology has a chance to increase There are no conflicts to declare.