To the Editor,Muller's ratchetConversely, recombination events with other homologous viruses can lead to emergence of novel properties, particularly during periods of replication and transmission in zoonotic hosts. Coronaviruses (CoVs) utilize a highly conserved replication strategy, making vaccines focusing on this vulnerability highly attractive. Subgenomic RNAs are transcribed by discontinuous transcription regulatory sequences, conserved nucleotides located near the 5′‐end of the genome. Recombination events are thus limited in their ability to propagate in the CoV family.Although RNA viruses typically have the ability to change rapidly (HIV and HCV) creating so‐called quasi‐species with profound differences in viral replicants due to the infidelity latent in the RNA polymerases, this appears to be less so with proofreading enzymes enabling restitution in the CoVs. Viral fitness is largely determined by the ability to propagate within the host and the host species, some reflecting that the most virulent strains are less successful from a broad host/pathogen perspective given that it ultimately limits viral spread. Genetic bottlenecks are created by interaction of viral variants and their host revealing a narrow range of genotypes successful over time.Genetic bottlenecks are likely to occur quite frequently with RNA‐based respiratory viruses since respiratory droplets often contain only one to two infectious particles per droplet.There may be evidence of bottleneck genetic selection with other viruses. In Ebola, mutations arose during the 2013 to 2016 outbreak which were postulated to increase or decrease virulence.During the severe acute respiratory syndrome (SARS) epidemic of 2003 to 2004, several stable mutations of SARS‐CoV were detected suggestive of bottleneck genetic selection. One of these mutations, a 29‐nucleotide deletion of ORF 8, arose early in the outbreak, was less pathogenic in cell culture, and was postulated to result in a loss of viral fitness due to a founder effect.Variation in SARS‐CoV‐2 spike proteins have been described, and one of these stable nonsynonymous substitutions, D614G, predicts for increased probability of protein glycosylation at a canonical NXS/T site at residue 616 of the viral spike protein, and correlates with differences in mortality and possibly transmissibility from coronavirus disease‐2019 (COVID‐19) observed comparing both coasts of the United States.Several of these mutations are predicted to increase or decrease protein glycosylation. A model of viral infection postulates that SARS‐CoV‐2 infection of the dendritic cell, the type II pneumocyte of the lung, and the endothelial cell is mediated through the DC/L‐SIGN/ACE2/CD209/CD147 complex found on these cells. Decreased glycosylation of the protein spike of SARS‐CoV‐2 should reduce viral binding to DCL‐SIGN and thus reduce viral uptake by these cells.There are some clues arising from studies of virulence of various SARS‐CoV‐2 isolates. Viral strains isolated from various patients with COVID‐19 in Wuhan can vary as much as 270‐fold in viral load and cytopathogenicity in Vero E6 culture.The involvement of Muller's ratchet in the pathogenesis and trajectory of the COVID‐19 pandemic should be explored. Examination of the distribution of nonsynonymous SARS‐COV‐2 mutations in symptomatic vs minimally symptomatic patients with COVID‐19 as the pandemic evolves could help support such involvement. Genomic analysis from SARS‐CoV‐2 isolates from ArizonaMuller's Ratchet should also be considered in developing vaccines for the disease, balancing natural decreases in virulence and limiting recombination events.The authors declare that there are no conflict of interests.


These results suggest that there are consistent correlations between aspects of behavior and morphology at large taxonomic scales , but the relationship between behavior and morphology depends largely on among‐clade differences and may be idiosyncratic at shallower scales . As a result, general relationships between behaviors and morphology may not be applicable when comparing close relatives.Understanding how co‐occurring species divide ecological space is a central issue in ecology. Functional traits have the potential to serve as a means for quantitatively assessing niche partitioning by different species based on their ecological attributes, such as morphology, behavior, or trophic habit. This enables testing ecological and evolutionary questions using functional traits at spatio‐temporal scales that are not feasible using traditional field methods. Both rapid evolutionary change and inter‐ and intraspecific competition, however, may limit the utility of morphological functional traits as indicators of how niches are partitioned. To address how behavior and morphology interact, we quantified foraging behavior of mixed‐species flocks of birds in the Solomon Islands to test whether behavior and morphology are correlated in these flocks. We find that foraging behavior is significantly correlated with morphological traits ( Understanding how co‐occurring species divide ecological space is a central issue in ecology. We demonstrate that morphology may reflect behavioral niche partitioning at large scales, but among islands in the Solomon Archipelago, shifts in avian morphology and behavior are decoupled. General relationships between behaviors and morphology may not be applicable when comparing close relatives. Classic studies of birds have tested niche partitioning hypotheses using detailed observations of behavior and various methods of quantifying ecological space to characterize the ecological niches of species , or complementary interactions among species in which interspecific differences provide benefits to group members , for example, include constituent species that mimic the babbler's plumage and call  were observed to follow the gregarious Gerygone species, apparently preying on disturbed insects . The limitations of observing birds in the rainforest canopy precluded collection of comprehensive lists of constituent species in each flock or precise estimates of relative abundances within flocks, but by pooling across all observations at a locality we have developed qualitative descriptions of flock composition for each island  was used to characterize foraging behavior. RA is an ordination method that can provide a statistical representation of ecological space, while simultaneously placing behavioral data within that space, and it is appropriate for use with categorical variables like our foraging data. This approach to characterizing foraging behavior has been used in previous studies to quantitatively describe foraging behavior for subsequent correlation with morphological data  and the pointedness of the bill (which we characterized as bill length/bill width). These traits have been associated with the degree to which species capture prey on the ground  and phylogenetic canonical correlation analysis , while others were less distinct . However, when phylogenetic relatedness was controlled for in the pCCA, this relationship was not significant . Similarly, relative tarsus length and bill pointedness were significantly positively related with the proportion of non‐aerial foraging .Results from the CCA indicated that morphology and foraging behavior of all taxa across islands are correlated, though this relationship is marginally significant . However, this correlation breaks down after accounting for phylogenetic relatedness (p = .66), suggesting that the relationship between morphology and behavior has an important phylogenetic component .Our results suggest that the relationship between behavior and morphology has an important phylogenetic component, and we do not find evidence that congeneric relationships between morphology and behavior are consistent with large‐scale patterns Figures  and 7. Wp = .01 and p = .002, respectively). This is in line with similar findings for distantly related ground foragers within the Tyrannidae in the New World tropics ; formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); visualization (lead); writing – original draft (lead); writing – review & editing (lead). Shahid Naeem: Conceptualization (supporting); resources (supporting); supervision (supporting); writing – review & editing (supporting). Benjamin M. Winger: Writing – review & editing (supporting). Joel Cracraft: Conceptualization (supporting); resources (supporting); supervision (supporting); writing – review & editing (supporting).Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.


Extracellular vesicles (EVs) are nanosized, membranous vesicles released by almost all types of cells. Extracellular vesicles can be classified into distinct subtypes according to their sizes, origins and functions. Extracellular vesicles play important roles in intercellular communication through the transfer of a wide spectrum of bioactive molecules, contributing to the regulation of diverse physiological and pathological processes. Recently, it has been established that EVs mediate foetal‐maternal communication across gestation. Abnormal changes in EVs have been reported to be critically involved in pregnancy‐related diseases. Moreover, EVs have shown great potential to serve as biomarkers for the diagnosis of pregnancy‐related diseases. In this review, we discussed about the roles of EVs in normal pregnancy and how changes in EVs led to complicated pregnancy with an emphasis on their values in predicting and monitoring of pregnancy‐related diseases. Extracellular vesicle‐derived heat shock protein family E member 1 (HSPE1) promotes Treg differentiation from CD4+ T cells and Treg cell expansion in vitro. Placental exosome‐derived miR‐499 is elevated in the first trimester in cows and could inhibit NF‐κB activation via Lin28B/let‐7 axis, thereby repressing inflammation response and forming an immune‐tolerant microenvironment in the uterus. miR‐499 inhibition leads to inflammation dysregulation and increased risk of pregnancy failure in vivo.Recent studies have highlighted a critical interaction between EVs and immune cells in modulation of pregnancy during which maternal immune system tolerates the growing foetus and maintains its normal functions.2.3To meet increased metabolic demands of the mother and foetus and to ensure adequate nutrients and oxygen supplies to the growing foetus, early stage of pregnancy requires sufficient spiral artery remodelling and physiological adaptations in the cardiovascular system.2.4Increasing evidence suggests that EVs are involved in metabolic homoeostasis.2.5Signals of foetus maturation probably induce inflammatory responses and thus prepare the uterus for delivery.3Abnormal changes of EV concentration and composition are involved in pregnancy‐related diseases, including pre‐eclampsia (PE), gestational diabetes mellitus (GDM), preterm birth (PTB) and other adverse pregnancy outcome Figure .3.1The dysregulation of the balance between pro‐ and anti‐inflammatory factors has been reported in complications of pregnancy.3.2Failures in endothelial functions and vasoconstriction responses increase the risk of vascular complications of pregnancy, especially PE.3.3Metabolism dysregulation is closely related to some pregnant complications, for instance, GDM and foetal overgrowth.4Extracellular vesicles have a tissue‐specific pattern and may reflect the status of source cells, which highlights their usefulness as indicators of cellular function and as biomarkers for various diseases. Increasing studies suggest that EV concentration and content are of great diagnostic utility for women at risk of pregnancy‐related diseases Table .4.1+NEP+ EVs is suggested as a promising biomarker of PE.+ EVs‐to‐nephrin+ EVs ratio is associated with renal injury in PE.Salomon et al4.2+ STBEVs in GDM maternal plasma is more than eightfold higher than that in normal pregnancies. Increased DPPIV biological activity is also demonstrated in STBEVs from GDM.The aberrant changes in placental microenvironment may affect the release and content of placenta‐derived EVs. The quantity of exosomes secreted from trophoblast cells cultured under high glucose condition is approximately three times higher than that cultured under normal glucose condition.4.3Ureaplasma and Veillonellaceae are more abundant in the urine of PTB women than normal deliveries.Extracellular vesicles have been suggested as indicators of preterm birth (PTB), with an advantage of non‐invasive isolation from maternal blood and other biological fluids.4.4+CD63+ exosomes to PLAP−CD63+ exosomes is used to describe the contribution of placental exosomes to the total. The ratio is positively correlated with the percentile of birth weight and displays a clear trend according to the severity of the disease. Thus, relative concentration of placental exosomes may be used as a marker for foetal growth. Rodosthenous et alIn a cohort study of pregnant women who give birth to small foetuses, Miranda et al5The current studies provide us a comprehensive understanding of EVs in normal and complicated pregnancy and an opportunity to develop novel methods for early and efficient diagnosis of pregnancy‐related diseases. Extracellular vesicles play diverse roles across the gestation such as embryo implantation, immunomodulation, vascular remodelling and metabolism adaptation by mediating foetal‐maternal crosstalk. The available data partially unravel the roles and underlying mechanisms of EVs in the physiology or pathophysiology of pregnancy. Extracellular vesicle‐derived bioactive molecules may explain their roles in regulating cellular functions and contribute to pregnancy‐related diseases. Moreover, the concentration and content of circulating EVs show potential diagnostic value as they may reflect condition changes, metabolism status, foetal growth and foetal maturation. The development of precise diagnosis may allow implementation of appropriate intervention, which would be helpful for reducing the harm to both mothers and foetus. However, there are still several problems that need to be addressed before EVs reach the clinic in human reproduction field. First, there are still no recognized common methods for the discrimination and quantification of different sub‐populations of EVs. It is necessary to establish stable and reliable methods to achieve rapid separation and automated analysis of EVs. Second, due to the difficulty of imitating long‐term effects in vitro and the lack of suitable cell and animal models, controversial and inconsistent results still exist in different studies. Moreover, human tissues usually only can be obtained after delivery and cannot accurately reflect the dynamic changes of markers during the whole pregnancy process. Therefore, more efforts should be devoted to giving an insight into the functions of EV in pregnancy and to apply EVs to the diagnosis, monitoring and treatment of pregnancy‐related diseases.The authors confirm that there are no conflicts of interest.JY Zhang and X Zhang contributed to the conception of the manuscript. JY Zhang, BY Fan and HB Li collected literature and draft the manuscript. WR Xu and X Zhang reviewed and made significant revisions to the manuscript. All authors read and approved the final manuscript.


Reports from countries severely hit by the COVID‐19 pandemic suggest a decline in acute coronary syndrome (ACS)‐related hospitalizations. The generalizability of this observation on ACS admissions and possible related causes in countries with low COVID‐19 incidence are not known.ACS admissions were reduced in a country spared by COVID‐19.We conducted a nationwide study on the incidence rates of ACS‐related admissions during a 6‐week period of the COVID‐19 outbreak and the corresponding control period in 2019 in Greece, a country with strict social measures, low COVID‐19 incidence, and no excess in mortality.P < .001) and for each ACS type . The decrease in STEMI admissions was stable throughout the COVID‐19 period , whereas there was a gradual decline in non‐STEMI/UA admissions  following the progressively stricter social measures. During the COVID‐19 period, patients admitted with ACS presented more frequently with left ventricular systolic impairment .ACS admissions in the COVID‐19 (n = 771) compared with the control (n = 1077) period were reduced overall (incidence rate ratio [IRR]: 0.72, We observed a reduction in ACS hospitalizations during the COVID‐19 outbreak in a country with strict social measures, low community transmission, and no excess in mortality. Medical care avoidance behavior is an important factor for these observations, while a true reduction of the ACS incidence due to self‐isolation/quarantining may have also played a role. Additionally, we assessed the impact of strict social measures on the observed ACS admission rate, and attempted to identify the presence of any cardiac collateral damage.22.1We conducted a multicenter, observational, nationwide study on patients admitted with ACS  to Greek public hospitals with PCI capability, including a primary PCI service. All consecutive patients admitted with a confirmed diagnosis of ACS at discharge were included in this study. Data were retrospectively collected for a 6‐week period from March 2 to April 12 during the COVID‐19 outbreak in 2020 and the corresponding 6‐week control period in 2019. The study complies with the Declaration of Helsinki and was approved by the local ethics committee at each hospital. Written approved consent was waived on the basis of the retrospective use of anonymized patient data.2.2The first confirmed COVID‐19 case in Greece was identified on 26 February 2020 and by 2 March 2020, there were only seven confirmed cases. Therefore, we identified 2 March 2020 as the beginning of the COVID‐19 period, and we studied the first 6 weeks of this period until 12 April 2020. During the 6‐week period of the study, the Greek government authorities progressively imposed several measures of social distancing which also contributed to an increase in public awareness: (a) 10 March 2020: lockdown of all schools and universities and recommendation of self‐confinement behavior; (b) 16 March 2020:80% of all business activities were locked down; and, ultimately, (c) 23 March 2020: drastic measures restricting the freedom of movement which continued until 3 May 2020. Until 13 April 2020 , there was a total of 2145 confirmed COVID‐19 cases and 99 deaths in Greece.To perform comparative analyses between the COVID‐19 study period and a period without exposure to COVID‐19, the corresponding period during the previous year (2 March 2019 to 12 April 2019) was used as control. Also, the study period was dichotomized to the time before  and after  the complete national lockdown, and comparative analyses were also performed for these two time periods during the COVID‐19 outbreak.2.3Categorical variables are presented as counts and percentages, and odds ratios (OR) with 95% confidence intervals (CI), and were compared using the chi‐square or Fisher's exact test as appropriate. Continuous variables included in the analysis did not have a normal distribution and are summarized as median and interquartile range (IQR); comparisons were made using the Mann‐Whitney test. Crude incidence rates (IR) for ACS admissions were calculated by dividing the number of cumulative events by the number of days for each time period. Incidence rate ratios (IRR), comparing the time periods , were calculated using the Poisson regression to model the number of ACS admissions per day. Regression analysis (linear and logarithmic) was used for testing a temporal trend of the relative change (incidence rate ratio) in ACS admissions during the 6‐week period. Linear regression analysis was also performed on the cumulative number of ACS admissions over the 42‐day time period separately for the study and control periods; analysis of covariance was used to compare the slopes of the two regression lines corresponding to the two periods. An alpha level of 0.05 was used to determine statistical significance unless otherwise stated for subgroup analyses; all statistical tests were 2‐tailed. All analyses were done with Stata 10.0  and SPSS 17.0 .3A total of 1848 ACS patients were included in the study (771 during the study period and 1077 during the control period). Detailed descriptive data of the population are presented in Table 3.1P < .001). Table The mean rate of ACS admissions in the study period 18.4/day) was significantly lower compared to the control period , whereas there was a gradual decline in NSTEMI/UA admissions   Figure .3.2P < .001).The cumulative number of ACS hospitalizations plotted over the 6‐week observation period (42 days) for 2020 and 2019, and their respective trend lines Figure , showed P = .002). The respective IRRs for the three ACS types . The mean rate of ACS admissions after the lockdown (16.3/day) was significantly lower compared to the period before the lockdown . The respective results within each ACS type indicate that this difference was primarily due to the subgroup of patients presenting with unstable angina (Table Patients admitted with ACS were less frequently above 65 years of age during the study period (44.2% of ACS admissions) compared to the control period . The separate results for each ACS type point out that this difference between the COVID‐19 and control period was particularly higher for STEMI patients and secondarily for NSTEMI patients, while there was no difference in UA patients (Table S2 in Appendix).LV systolic impairment (defined as ejection fraction <40%) at ACS presentation  was registered more frequently during the COVID‐19 period compared to the control period . The proportion of STEMI patients undergoing primary PCI during the COVID‐19 period was lower compared to the control period  reflecting the numerically higher frequency of treatment with thrombolysis at the initial stage of STEMI presentation. Similarly, the proportion of patients with NSTEMI or UA undergoing PCI during the COVID‐19 period was also lower .Overall, patients presenting with ACS underwent PCI less frequently during the COVID‐19 period compared to the control period . There was no difference in the rate of cardiac deaths between the two periods , while the rates of in‐hospital repeat MI  and stent thrombosis  were numerically higher during the COVID‐19 study period compared to the control one without reaching statistical significance.In all ACS patients, there was a total of 54 in‐hospital deaths (3%) similarly distributed in the two periods  there was a significant reduction in the rate of all ACS‐related admissions which was evident for all three ACS types  in the COVID‐19 compared with the control period; (b) the decline of STEMI admissions was relatively stable throughout the COVID‐19 period, while the number of NSTEMI/UA admissions declined even more strikingly after the complete national lockdown; (c) the proportion of all ACS patients undergoing PCI as well as the proportion of STEMI patients treated with primary PCI was lower during the COVID‐19 period.The overall reduction of ACS‐related admissions (28%) during the COVID‐19 outbreak in Greece is in line with contemporary reports about a 26% reduction in ACS hospitalizations in Northern Italy,Multiple factors may contribute to our observations regarding the decline in ACS admissions. First, this reduction may reflect a decreased contact of patients with the healthcare system. Fear among the public for potential exposure to SARS‐CoV‐2 and getting infected in a medical facility along with strict guidelines for self‐isolation may have inadvertently led many patients to avoid or delay seeking medical help. Supporting observations for this factor include the following. (a) There was a pronounced decline in the rate of admissions observed after the complete national lockdown only for ACS types with less severe clinical presentation/symptoms ; similar findings were observed during the national lockdown period in Northern Italy. (b) Older patients with an ACS (mainly UA) presented less frequently to the hospitals during the COVID‐19 period; older patients were considered from the start of the outbreak as one of the population groups at highest risk from COVID‐19 and were urged early by mass media and government authorities to avoid social contact and be self‐confined. (c) During the COVID‐19 outbreak, ACS patients presented more frequently with LV systolic impairment, which may serve as a surrogate marker for delayed MI presentation, and is supported by longer times from symptom onset to first medical contact observed for MI patients in other countries.An alternative cause for the decline in the rate of ACS hospitalizations is a true reduction in the incidence of ACS. This could be attributed to a lack of environmental triggers for MI due to the large‐scale measures of public and financial lockdown. Such measures decrease exposure to traffic, air pollution, and other respiratory infections , all of which have been associated with acute MI.Apart from the decline in ACS‐related admissions, we observed changes regarding the in‐hospital management of ACS with a relatively lower frequency of patients undergoing PCI for all ACS, including primary PCI for STEMI patients, which reflects a higher proportion of patients managed medically and a relatively higher use rate of thrombolysis in STEMI cases during the COVID‐19 pandemic. This is in line with reports from other countries about small increases in pharmacological thrombolysis and a decrease in the rate of PCI for NSTEMI patients.The afore‐mentioned observations and speculations have important inferences for public health, government policies regarding the management of pandemics, and possibly social structure influencing triggering factors for ACS. Epidemiological data show that countries with low COVID‐19 incidence did not have a substantial change in mortality, whereas countries with high COVID‐19 incidence presented an excessive deviation in mortality from the expected level in most cases. In the latter, excessive deaths could be caused both by COVID‐19 and by collateral damage, such as in the case of ACS patients who may have avoided or did not receive proper treatment. In Northern Italy, an increase in mortality observed during the COVID‐19 outbreak could not be fully explained by COVID‐19 cases alone.4.1The data presented are observational and no definitive conclusions can be drawn about causality. This is a retrospective study with all shortcomings associated with such an approach including the lack of additional variables of interest, which were not readily available. However, our study gathered comparative data over a 6‐week period from the huge majority of hospitals with primary PCI services in the country, thereby providing representative information on ACS‐related hospitalizations.5We observed a decline in ACS‐related hospitalizations during the COVID‐19 outbreak in a country with low penetrance of SARS‐CoV‐2 virus, strict measures of social restrictions and no substantial increase in overall population mortality. Our findings provide indirect evidence that medical care avoidance behavior among ACS patients is an important factor for these observations, while a true reduction of the ACS incidence due to self‐isolation/quarantining, and thus, a lack of environmental triggers for ACS, cannot be excluded. Further research is needed to clarify these factors.The authors declare no potential conflict of interest.AppendixS1. Supporting Information.Click here for additional data file.


Optimal management of patients with upper rectal cancer remains unclear. Partial mesorectal excision (PME) without neoadjuvant therapy is currently advocated for the majority of patients. Recent studies, however, reported a high risk of local recurrence and suboptimal surgery. The aim of this study was to evaluate the effects of a quality assurance initiative with postoperative MRI to improve outcomes in these patients.Patients who underwent mesorectal excision with curative intent for rectal cancer in 2007–2013 were included. Postoperative MRI of the pelvis was performed 1 year after surgery. In 2011, a multidisciplinary workshop with focus on extent and completeness of surgery was held for training surgeons, pathologists and radiologists involved in treatment planning. Images of residual mesorectum and histopathological reports were reviewed with regard to the distal resection margin. Local recurrence after a minimum of 3 years' follow‐up was compared between two cohorts from 2007–2010 and 2011–2013.P = 0·012). After the workshop, fewer patients with cancer of the upper rectum were selected to have PME , and fewer patients who underwent PME had an insufficient distal resection margin .A total of 627 patients were included; postoperative MRI of the pelvis was done in 381 patients. The 3‐year actuarial local recurrence rate in patients with upper rectal cancer improved from 12·9 to 5·0 per cent (Quality assessment of surgical practice may have a major impact on oncological outcome after surgery for upper rectal cancer. With a local quality assurance programme, significant improvement in local recurrence rates for cancer of the upper rectum was achieved by focusing on preoperative selection of type of surgery and the extent and completeness of mesorectal excision.'Multidisciplinary training program helped to improve local recurrence rates' Management of rectal cancer has improved over recent decades, primarily by the standardization and improvement of surgical procedure. Whereas total mesorectal excision (TME) or abdominoperineal excision (APE) is optimal for mid and low rectal cancer, there has been little focus on the optimal management of upper rectal cancer. Tumours of the upper rectum  may not require TME, and may be managed optimally by perpendicular transection of the mesorectum at least 5 cm below the lower edge of the tumour. Partial mesorectal excision (PME) is currently advocated for the majority of tumours of the upper rectum, based on the rationale that preservation of a distal part of the rectum and mesorectum will result in better long‐term functional outcome and fewer postoperative complications, and assumed similar oncological safetyStandardization and quality assurance of mesorectal excision by training and pathological audit were implemented in the major trials to ensure that optimal surgery was performed. It is important, however, that outside the setting of clinical trials standardization and assurance of best quality surgery are also sustained in routine clinical practice. By using postoperative MRI it is possible to assess the extent and completeness of mesorectal excision by focusing on the tissue left behind, as opposed to tissue removedA concerningly high rate of local recurrence (13·5 per cent) in patients with upper rectal cancer after PME has been reported previouslyThe aim of this study was to assess the effect of a multidisciplinary‐initiated quality assurance programme in patients treated for upper rectal cancer with regard to local recurrence, type of surgery, residual mesorectum and distal resection margin.Consecutive patients with rectal adenocarcinoma, defined as cancer 15 cm or less from the anal verge on rigid proctoscopy, who underwent mesorectal excision with curative intent at Aarhus University Hospital or Regional Hospital Randers, two hospitals in Denmark, from August 2007 to 2013 were included in the study cohort. All patients were discussed at a multidisciplinary team (MDT) conference based on preoperative MRI of the pelvis (if applicable), CT of the chest and abdomen, and a clinical examination with rigid proctoscopy.Danish guidelinesPatients who had surgery without signs of distant metastasis and with tumours considered to be resectable with a clear margin at preoperative evaluation or after neoadjuvant therapy were classified as having treatment with curative intent, irrespective of the pathological circumferential resection margin (CRM) involvement at definitive histopathological examination. Type of surgery was determined from the surgical reports. Approval for the study was obtained from the Local Committee for Health Research Ethics and the Danish Data Protection Agency (2009‐41‐4056).The length of the distal resection margin (DRM) was measured in all rectal cancer specimens at histopathological examination. The length of the DRM was measured on the fixed specimen after sectioning in 5‐mm slices, as the distance between the luminal lower border of the tumour and the distal cut edge. A DRM of less than 3·5 cm in PME resections was evaluated as insufficient based on findings of a previous studyTumour height had been documented by both rigid proctoscopy at preoperative evaluation and on preoperative MRI. The height measured by rigid proctoscopy was set as the method of reference. On MRI, tumour height was measured as the distance between the lower border of the subcutaneous part of the external sphincter, reflecting the anal verge, and the most distal part of the luminal tumour.Generally, patients were included in a follow‐up regimen with outpatient visits at 3, 6, 12, 18, 24 and 36 months after surgery. At each visit, a clinical examination and rigid proctoscopy (not applicable in patients who had APE) were performed. CT of the chest and abdomen was performed according to the protocol for the COLOFOL trialConsecutive patients were invited for postoperative MRI of the pelvis. Patients with disseminated disease, previous diagnosis of local recurrence, death at inclusion or contraindication to MRI were not eligible. Patients had MRI at least 6 months from the time of primary surgery.MRI was performed using a Magnetom Avanto 1·5‐Tesla MRI Scanner® . Sagittal, axial and coronal T2‐weighted turbo spin‐echo images were obtained in addition to a sagittal short T1‐inversion recovery of the bony pelvis and a sagittal T23D sequence of the smaller pelvis. The radiologist was blinded to the pathological assessment and all clinical data, with the exception of preoperative MRI findings and type of surgery. Evaluation of the postoperative MRI included assessment for the presence, localization and size of residual mesorectum, level of anastomosis and detection of local recurrence. The same radiologist evaluated all radiological examinations together with the first author for consensus. Mesorectal fatty tissue with a discernible tissue interface of fibrosis, which separates the mesorectum from the mesocolon, was considered a sign of residual mesorectum and categorized as described previouslyQuality assessment of surgery in the two departments has been performed since 2007 by postoperative MRI and histopathological examination. In 2011, an MDT‐initiated workshop with focus on the extent and completeness of mesorectal excision was held with participation of all involved surgeons, pathologists and radiologists. A specific focus was on upper rectal cancer and PME based on postoperative MRI findings between 2007 and 2010. A description of the procedure with comparison of preoperative and postoperative images together with histopathological section was presented. Documentation of inadvertent residual mesorectum following mesorectal excision, length of DRM, histopathological examination, and comparison between tumour height on rigid proctoscopy and preoperative MRI were reviewed and discussed in a plenary session.After the workshop, quality assessment of treatment and surgery continued by postoperative MRI and histopathological examination according to the same protocol.t test and the χ2 test for continuous and categorical data respectively. Local recurrence rates were estimated using Kaplan–Meier actuarial methods between the two time periods, 2007–2010 and 2011–2013. The log rank test was used for comparisons, as well as for subanalysis in patients with tumours of the upper rectum. Time to local recurrence was measured from the date of primary surgery to the date of diagnosis of local recurrence. Patients without local recurrence were censored on the date of their last outpatient visit or upon death.Demographic and patient characteristics were summarized using median (range) values and percentages, with comparisons performed using Student's P < 0·050 was considered to represent statistical significance.Statistical analyses were performed using Stata® version 12  and SigmaPlot® . Table versus 8·5 per cent in 2007–2010; P < 0·001). Of the 627 patients, 381 (60·6 per cent) had postoperative MRI performed a median of 12 months after primary surgery.A total of 627 patients underwent curative resection with mesorectal excision for rectal adenocarcinoma between 2007 and 2013. Clinical characteristics of these patients according to the two time periods are shown in P = 0·156) .After a median follow‐up of 36 range 0·2–107) months, 42 patients developed local recurrence. The overall 3‐year actuarial local recurrence rates in 2007–2010 and 2011–2013 were 7·3 (95 per cent c.i. 4·9 to 10·9) and 4·6 (2·5 to 8·4) per cent respectively (–107 montP = 0·012)  . After the workshop, fewer patients with upper rectal cancer underwent PME compared with TME; the rate of TME increased from 9·2 per cent in 2007–2010 to 19·8 per cent in 2011–2013 (P = 0·023). No patient who had TME for cancer of the upper rectum developed local recurrence . No patient treated with PME developed local recurrence when a DRM of at least 3·5 cm was achieved . This improved in the later cohort, with a decrease from 61·7 per cent in 2007–2010 to 31 per cent in 2011–2013 (P = 0·458). The mean volume of inadvertent residual mesorectum was 8·03 and 5·74 cm3 in 2007–2010 and 2011–2013 respectively (P = 0·250).Inadvertent residual mesorectum was identified in 163 (42·8 per cent) of the 381 patients who had postoperative MRI. In patients who underwent PME, inadvertent residual mesorectum was detected in 60·4 per cent (84 of 139). This did not differ between 2007–2010 and 2011–2013: 60 per cent (50 of 84) and 62 per cent (34 of 55) respectively (Based on the preoperative tumour status (mrT), T4 disease was overrepresented in patients with local recurrence who underwent PME or APE. All pT4 tumours of the upper rectum were due to local peritoneal involvement (pT4a).In 2007–2010 and 2011–2013, long‐course preoperative radiochemotherapy was administered in 24·6 per cent (31 of 126) and 22·5 per cent (25 of 111) respectively of patients who had TME, and in 62·4 per cent (68 of 109) and 51 per cent (37 of 72) of those who underwent APE. In patients who had PME, neoadjuvant therapy was not used in 2007–2010, whereas it was employed in 4 per cent (3 of 81) of patients who underwent PME in 2011–2013.This study has shown a significant reduction in the risk of local recurrence after a local MDT‐directed quality assurance programme in patients with cancer of the upper rectum. A previous reportTo obtain and maintain improvements in quality of rectal cancer surgery, it may be necessary to consolidate the training by organizing continuous workshops with a focus on difficulties in the current treatment of rectal cancer, in order to sustain best quality treatment in routine clinical practice. By using postoperative MRI, it is possible to assess the extent and completeness of mesorectal excision, to highlight difficulties in current management of upper rectal cancer. One of the great hallmarks of MDT collaboration is quality assurance and internal audit of the quality of treatment performed.For cancer of the upper rectum, it remains unsettled as to whether TME is necessary, or whether PME is adequate. Unfortunately, the major studies investigating the initial improvements following the introduction of TME have not specifically distinguished the type of procedure for upper rectal cancer, hindering comparisonet al.Based on data from the Stockholm Colorectal Cancer Study Group, Syk and colleaguesThe risk of local recurrence in patients with upper rectal cancer treated in 2007–2010 corresponds to the results presented by Syk and colleaguesAn effort to detect suboptimal surgery with postoperative imaging was attempted before the advent of modern MRI. By using postoperative angiography of the mesenteric artery, Hohenberger and co‐workerset al.In a recent educational tutorial by Kuzu Kusters and co‐workersNo difference in the reported prevalence of inadvertent residual mesorectum on postoperative MRI was observed in the present study after the workshop, despite the observed improvement in local recurrence. Small amounts of residual mesorectum in sufficient clearance of the primary tumour may not be associated with an increased risk of local recurrence, similar to pathology reports that an intramesorectal excision may not be associated with an increased risk of local recurrence compared with that following complete mesorectal excisionet al.There is ongoing controversy as to whether patients with cancer of the upper rectum will benefit from neoadjuvant therapy. In Denmark, only patients with unresectable cancer of the upper rectum are considered for neoadjuvant therapy, as opposed to those with advanced mid or low rectal cancer. Large population‐based studies from Sweden have reported that more than 60 per cent of patients with rectal cancer received neoadjuvant therapy, including 43–58 per cent of patients with tumours located in the upper rectum, and more than 40 per cent of those with T1–2 tumoursThe characterization of rectal cancer into high, mid and low lesions is traditionally measured from the anal verge using a rigid proctoscope. Although this measurement is mandatory in Denmark, it may vary depending on the surgeon performing the examination, the patient and the methodIn their 2016 annual reportIn the present authors' opinion, the height of the tumour is assessed most reliably by preoperative MRI, and can be used to determine whether PME or TME is preferable. By using the Delphi method, the European Registration of Cancer Careet al.Limitations of the present study include the differences in the baseline characteristics between the patient cohorts in the two time periods, and that the implementation of laparoscopic surgery in the second period may also have potentially influenced outcomes. All MRI examinations were evaluated by the same radiologist in consensus with the first author. No previous analyses on interobserver variability have been reported. In a recent study by Veltcamp Helbach A national audit of the quality of surgery in Denmark is currently being conducted including postoperative MRI, and a training programme for colorectal surgeons with a specific focus on surgery for upper rectal cancer is planned.


Human papillomavirus (HPV) infection is the most common cause of cervical cancer and can be prevented with vaccination, but HPV vaccination rates remain low. An intervention to improve health care provider communication about vaccination has been shown to increase HPV vaccination rates in an initial trial in Colorado, where about 160 cases of cervical cancer are diagnosed each year.Census data were combined with Colorado cancer and immunization registry data to identify clinics in locations that would most benefit from implementation of this intervention to improve HPV vaccination rates. ArcGIS Pro was used to map cervical cancer incidence, immunization rates, population data, and location of clinics participating in practice‐based research networks (PBRNs). Results from the provider communication intervention trial and published estimates of the number needed to vaccinate to prevent a case of cervical cancer were used to predict the number of cervical cancer cases prevented based on increased vaccination due to the intervention.Ninety‐eight Colorado PBRN clinics were analyzed. For the 10 clinics with the highest predicted number of cervical cancer cases prevented, 5218 additional patients would be vaccinated and 43 cervical cancer cases prevented with implementation of the intervention. If implemented in all 98 clinics, the intervention would lead to 20 490 additional patients vaccinated (range 7‐658/clinic) and 171 cases of cervical cancer prevented (range 0.05‐5.48/clinic).Geographic data from cancer and immunization registries can inform the dissemination of evidence‐based practices like the provider communication intervention for HPV vaccination to maximize impact on public health. A model predicting the number of cervical cancer cases prevented with implementation of an evidence‐based intervention to increase HPV vaccination was developed using cancer and immunization registry data and published estimates of the number needed to vaccinate to prevent one case of cervical cancer. Population, cervical cancer incidence, immunization rates, and clinic locations across Colorado were analyzed to identify clinics that would benefit most from the intervention. This analysis demonstrates how spatial data from existing registries can identify clinics with the highest predicted number of cervical cancer cases prevented, thus maximizing the public health impact of an HPV vaccination intervention. Eight practices were included in the intervention group, and each of the practices in the study was concentrated around the most populous metropolitan area of Colorado. The intervention included HPV fact sheets, a parent education website, pictures of diseases caused by HPV, a decision aid for the HPV vaccination, and HPV vaccine communication training for health care professionals. Adolescents in the intervention practices had significantly higher odds of HPV vaccine series initiation  and completion  than those in the control practices (a 9.5—absolute percentage point increase in HPV vaccine series initiation and a 4.4—absolute percentage point increase in HPV vaccine series completion in intervention practices).2.2There were 174 clinics affiliated with Colorado PBRNs that were considered for this study. These PBRN clinics are situated in both rural and urban areas around Colorado , the state's population‐based cancer registry that includes the geocoded residence of cases at the time of diagnosis. All Colorado residents who are diagnosed with cancer are included in the database, regardless of whether or not they receive their diagnosis in Colorado.Incidence density maps were created using ArcMap 10.3.1 to show the distribution of cervical cancer cases in Colorado based on the residence location of each case.2.5Zip‐code level HPV vaccination data from 2017 was obtained from the Colorado Immunization Information System (CIIS), collected by the Colorado Department of Public Health and Environment. These data included females aged 11‐17 years that had received at least one dose of the HPV vaccination. The number of vaccinated females aged 11‐17 years in intersecting zip codes of the 2‐mile buffers was divided by the total number of females age 11‐17 in the zip codes, to create baseline vaccination rates for the clinics. Zip‐code level vaccination rates were used to estimate clinic immunization rates because obtaining clinic‐specific immunization data from all PBRN clinics was not feasible with existing data and privacy infrastructure limitations. A predicted change in vaccination rate post‐intervention was calculated for PBRN clinics using a linear regression model derived from the initial HPV provider communication intervention study, looking at the association between baseline clinic vaccination rates and the change in vaccination rate from pre‐ to post‐intervention.2.6The estimated NNV was based off a literature review, where a systematic review study was used to determine this value.2.7PI) was first calculated using the population eligible for vaccination and the baseline and post‐intervention vaccination rates. The cervical cancer cases we would expect to prevent with the intervention was then calculated by dividing the NVPI by the estimated NNV. The expected post‐intervention incidence was calculated using the baseline number of cases, the cases prevented with the intervention and the population at risk. Lastly, the preventable fraction was calculated using the baseline cervical cancer incidence and the expected post‐intervention cervical cancer incidence.The preventable fraction of cervical cancer cases was calculated for each clinic and was defined as the proportion of cervical cancer that we would expect to prevent in the population at risk by increasing HPV vaccination rates with the provider communication intervention. The number of people that we would expect to vaccinate with the intervention  the 9‐26 year age range of eligibility for routine HPV vaccination. If increased vaccination rates are sustained after the intervention (which remains to be studied), future age cohorts would also benefit from increased vaccination rates and contribute to the predicted number of cervical cancer cases prevented in the future.The methods presented in this study illustrate one way to use cancer registry and immunization registry data to target vaccination interventions for primary prevention of HPV‐related cancer. Data from of cancer registries in the US and around the world have contributed tremendously to the understanding of epidemiologic trends and helped to identify factors associated with cancer diagnosis and outcomes. Data from immunization registries are often used to identify areas or populations with low vaccination rates or to assess changes in vaccination rates after interventions at the practice, public health, or policy level. By combining geographic information from both the cancer registry and the immunization registry, we used existing data sources to identify locations where high HPV‐related cancer incidence and low HPV vaccination rates overlapped. Each state in the US has its own central cancer registrySpatial analysis of vaccination and cervical cancer rates can inform an approach focused on the population at risk, however, identifying the population at risk is only a part of ensuring optimal dissemination of HPV vaccination interventions. Additional study is needed to identify other factors that may predict the success of the interventions like the HPV provider communication intervention in various practice settings such as provider type, practice size, and organizational factors. We are currently using qualitative methods to conduct pre‐implementation assessments among providers from PBRN clinics in at‐risk locations.4.1There were several limitations in this study that could have an impact on the results or the generalizability of the study. First, it is difficult to predict how far individuals are willing to travel to seek primary care. A 2‐mile buffer was drawn around PBRN clinics for these analyses which may not reflect the true population of patients seen at each clinic, particularly in rural areas where people often travel long distances to seek care. For the PBRN locations in urban areas, a 2‐mile buffer zone helped to decrease overlap in clinic catchment areas. Because we do not have clinic‐level data about the location of residence for each clinic's actual patient population, we used the same size buffer for every clinic. We assumed an even distribution of populations within block groups, however if population density varies within block groups, then the estimated population at risk within the 2‐mile buffer zones may not be accurate. Some clinic estimates for the preventable fraction of cervical cancer cases exceeded 100%, implying that we did not have an accurate estimate of the population at risk surrounding those clinics. We did not know the true population at risk surrounding each clinic, nor could we without conducting a detailed survey of each clinic's patient population. Our approach still identifies those clinics most likely to benefit from receiving the intervention, even if the absolute value of the preventable fraction of cervical cancer cases is over‐estimated. An additional limitation of our approach is that we did not predict the impact of adolescents changing their location of residence after vaccination, which could have varying effects on the number of cervical cancer cases prevented in each location.Zip code level vaccination rates may not reflect the population truly attending a specific clinic in that location. Clinics might draw their patients from farther afield than their local zip code or from only one part of their local zip code that might have a different vaccination rate than the zip code average represents. Use of zip code level vaccination data is imperfect but allows for use of population‐level data from an existing database (CIIS) rather than collecting data separately for each clinic. The estimated NNV for HPV vaccine to prevent one case of cervical cancer is reported across a broad range in the published literature (120‐324) and does not account for differences in vaccine effectiveness based on age at vaccination. The variability in this value may have impacted our analyses by biasing the results toward finding a higher predicted number of cervical cancer cases prevented because we chose the lower estimated NNV, however this bias would be unlikely to change the relative distribution of cases prevented across the geographic areas analyzed. In addition, we used rates of HPV vaccine initiation rather than completion for our analyses. Published estimates of NNV to prevent cervical cancer are based upon HPV vaccine completion; however, emerging data may support the effectiveness of single‐dose HPV vaccination,The analysis presented here looked at predicted impact of an intervention to increase HPV vaccination rates among populations ages 9‐26, however the initial HPV provider communication intervention was not tested beyond the adolescent period and the baseline vaccination rates examined were for adolescents 11‐17 years. It is unknown whether the gains of the HPV provider communication intervention would also be seen among young adults. This study focused on cervical cancer and HPV vaccination among females to demonstrate the modeling methods. In future work, we will incorporate vaccination rates and HPV‐related cancers among males. Because the referenced intervention was based at the clinic level without specific focus on patient‐level socioeconomic characteristics, we did not assess for such factors in this geospatial model. We plan to assess socioeconomic factors using census data in future work. Generalizability of our findings may be limited as PBRN clinics may differ from primary care clinics that are not part of a research network.HPV vaccination is a powerful and underutilized tool for preventing cervical cancer. We present an example of how geographic data from cancer and immunization registries can be used to identify clinics with the highest predicted number of cervical cancer cases prevented if an intervention was to be implemented. These methods should be applied in planning the dissemination and implementation of interventions like the provider communication intervention for HPV vaccination in order to maximize the predicted impact on public health.Amanda Dempsey serves on advisory boards for Merck, Pfizer and Sanofi. She did not receive research support from these companies. The other authors have no potential conflicts of interest.Jessica Cataldi: Data curation, project administration, writing—original draft, and writing—review and editing. Marian Håbesland: conceptualization, data curation, formal analysis, visualization, writing—original draft, and writing—review and editing. Amy Anderson‐Mellies: data curation, formal analysis, investigation, methodology, software, validation, visualization, writing—original draft, and writing—review and editing. Amanda Dempsey: Conceptualization, methodology, writing—review and editing. Myles Cockburn: Conceptualization, methodology, resources, supervision, writing—review and editing. In addition, all authors have reviewed the submitted manuscript, provided final approval of this version, and agree to be accountable for all aspects of the work.


Liquid biopsies of cancer via single‐cell molecular profiling of circulating tumor cells (CTCs) are hampered by the lack of ideal CTC markers. In this study, it is reported that TPN, a bioprobe with aggregation‐induced emission (AIE) activity is capable of distinguishing various tumor cells from blood leukocytes based on the difference in cell mitochondria. TPN is a cell‐permeant live‐cell stain that has little effect on cell viability and integrity, enabling single‐cell DNA/RNA analysis with improved efficiency compared with traditional antibody‐based methods. Using TPN labeling, CTCs and CTC cluster are detected in the blood from patients with lung or liver cancer. The capability of TPN to identify rare tumor cells in the malignant pleural effusion samples is also demonstrated. Furthermore, RNA sequencing of single lung CTC identified by TPN is successfully performed. The findings presented here provide an antibody‐free, low‐cost, and nondisruptive approach for detection and genomic characterization of viable tumor cells based on a mitochondria‐targeting AIE luminogen. It might serve as a new tool for monitoring of genomics dynamic of tumor and unraveling the mechanisms of tumor metastasis. A mitochondria‐targeting bioprobe with aggregation‐induced emission (AIE) activity for detecting rare tumor cells in the blood and pleural effusion from cancer patients is presented. The cell‐permeant, low toxic bioprobe enables genomic analysis of single tumor cells with improved performance, and can serve as a promising tool for liquid biopsy to obtain cancer genetics. Metastasis is the hallmark of malignancies and the leading cause of cancer‐related deaths.Precision oncology aims to improve patient prognosis through molecular profiling of tumors. A particular advantage of CTCs compared with other cancer biomarkers is that they carry the whole genome from primary tumor.Mitochondria are essential organelles that are responsible for energy production in eukaryotes.Here, we report a luminogen with aggregation‐induced emission (AIE) activity named TPN for noninvasive identification of tumor cells among blood cells. TPN can label mitochondria of multiple cancer cell lines with significantly higher fluorescence than that of white blood cells. Compared with traditional identification methods for CTCs marker, we show that TPN has little effect on cell quality and thus allows DNA or RNA to be analyzed on single‐cell level with improved performance. Using samples from cancer patients, we also demonstrate the capability of TPN to identify rare tumor cells in blood as well as malignant pleural effusion.FigureTPN consists of a prototypical AIE unit, tetraphenylethene (TPE), and a pyridinium group Figure1a. It −/CD45+) were weakly emissive. In contrast, intense yellow fluorescence could be found from the reticulum‐like mitochondria within H1975 cells (CMFDA+/CD45−), which can be well distinguished from the leukocytes. Based on the marked difference in fluorescence, we identified 94.0 ± 3.9% (mean ± standard deviation (SD)) of the spiked H1975 cells. In addition, high recovery  was also achieved using liver cancer cells (HepG2) as model . These results indicate that TPN is capable of labeling tumor cells with high efficiency and selectivity. We also used MitoTracker Green (MTG), a well‐known commercial mitochondrial dye for comparison. Analysis by flow cytometry shows that the mean fluorescent signal of A549 cells is just 1.2 times higher than that of leukocytes , which is significantly lower than that of the TPN (4.1 times). We observed that tumor cells spiked into leukocytes exhibit faint fluorescence after MTG labeling. Compared with TPN staining, the difference in MTG signal between leukocytes and tumor cells is too small to distinguish . Since the working concentration of MTG in this experiment is relatively low , the accompanying low dynamic range may attribute to the indistinctive difference in cellular fluorescence. Further increasing the concentration of these probes may result in the aggregation‐caused quenching (ACQ) effect and nonspecific binding.−6m) of TPN was used and the emission of TPN could be intensified along with the increased accumulation of dye in mitochondria of tumor cells. This could be important to its better selectivity to tumor cells.As flow cytometry does not possess the sensitivity to analyze rare cells in blood, we next spiked a small number of tumor cells in leukocytes to mimic the real samples. A serial of known numbers 5–110) of lung cancer cells (H1975) were prelabeled with 5‐chloromethylfluorescein diacetate (CMFDA), a green‐emissive dye, followed by spiking into leukocytes and costained with TPN and anti‐CD45 antibodies (leukocyte marker). Cells that emitted bright yellow fluorescence were considered as tumor cells and verified by the existence of CMFDA emission. As shown in Figure 10 of lunWe next sought to evaluate the effect of TPN labeling on downstream analysis. Propidium iodide (PI), a stain for dead cell, was used to examine cell viability after TPN labeling . We observed that most cells from various cell lines remained viable after TPN and PI staining , indicative of the low toxicity of TPN.FigureTo further test whether TPN would affect molecular analysis at the single‐cell level, we evaluated the performance of TPN labeling in downstream analysis and used traditional CKs marker that were stained by immunofluorescence as a comparison Figure2a. Sin−1  can be detected from the CKs‐labeled cell  and liver cancer (n = 22) . CTCs were first enriched by a spiral microfluidic‐based automated cell retrieval system (ClearCell FX),n = 12). Nucleated cell emits strong yellow fluorescence, absent staining for CD45 (TPN+/CD45−/Hoechst+) with a high nuclear/cytoplasm ratio but without a segmented nucleus (characteristics of the granulocytes) was enumerated as a putative CTC. Based on this criteria, CTCs were detected in 53 of 68 patients (77.9%) with lung cancer  in both specimens. In contrast, no putative cells were found in three pleural effusion samples caused by benign diseases , demonstrating the high potential of TPN as a new tool for malignant pleural effusion diagnosis.We then evaluated the performance of TPN with clinical samples. Whole blood samples were collected from patients with advanced lung cancer  for library construction and transcriptome sequencing. The sequencing data got scores of higher than 32 assessed by Phred quality scoring , indicative of its reliability. The sequencing data show that among the most abundantly expressed transcripts, we may find genes of MALAT1, GSTO1, CTTN, THBS1, and SPARC that have been reported to be correlated with tumorigenesis,FigureThe superior performance of TPN prompted us to test its feasibility in single CTC RNA sequencing that relies heavily on cell quality. We micromanipulated single TPNIn summary, we have developed a mitochondria‐targeting AIE bioprobe (TPN) for detection of rare tumor cells among normal cells. After incubation of TPN, tumor cells can be well‐distinguished from white blood cells with high‐contrast because they exhibited stronger TPN fluorescence than human leukocytes. This may due to the metabolically active tumor cells have more mitochondria with elevated mitochondrial membrane potentialExperimental details can be found in the Supporting Information.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.In March 2020, with lockdown due to the coronavirus pandemic underway, the Francis Crick Institute (the Crick) regeared its research laboratories into clinical testing facilities. Two pipelines were established, one for polymerase chain reaction and the other for Serology. This article discusses the Cricks Flow Cytometry Science Technology Platform (Flow STP) role in setting up the Serology pipeline. Pipeline here referring to the overarching processes in place to facilitate the receipt of human sera through to a SARs‐CoV‐2 enzyme‐linked immunosorbent assay result. We examine the challenges that had to be overcome by a research laboratory to incorporate clinical diagnostics and the processes by which this was achieved. It describes the governance required to run the service, the design of the standard operating procedures (SOPs) and pipeline, the setting up of the assay, the validation required to show the robustness of the pipeline and reporting the results of the assay. Finally, as the lockdown started to ease in June 2020, it examines how this new service affects the daily running of the Flow STP. © 2020 The Authors.  At the beginning of February 2020, the COVID‐19 outbreak in the UK gathered pace and it seemed highly probable that the United Kingdom would follow similar lockdown restriction policies seen in other European countries. The Crick’s Flow Cytometry Science Technology Platform (Flow STP) helped prepare scientists to finish current experiments and store experimental material during lockdown to enable an efficient restart upon the eventual lifting of the restrictions. During this time safety measures were introduced into the Flow STP, including social distancing, that directly reduced the number of instruments available for use. When lockdown was announced in the United Kingdom on the 23rd March 2020 the total number of staff allowed into the Crick was reduced to a core group of key workers. The Flow STP was given key worker status and operated to provide flow cytometry to Crick scientists whose sole focus was now COVID‐19.In the lead‐up to lockdown communications were sent to scientists to highlight the possible implications of the pandemic on Flow STP operation. There were significant changes in the way the Flow STP operated due to the challenges faced outlined in Table In early May 2020 the Crick prepared to support testing during the pandemic by establishing the Crick COVID‐19 Consortium that successfully developed a diagnostic polymerase chain reaction (PCR) pipeline  laboratory was created, housing a ZE5™  and LSR Fortessa™ , for analysis of serological samples. A second CL2 laboratory was created for ELISA testing.To operate as a clinical diagnostic lab specific standards must be attained. The United Kingdom Accreditation Service (UKAS) is a government‐recognized body that provides certified testing, inspection, and calibration services together with an oversight function. UKAS requires medical laboratories to have International Organization for Standardization (ISO) 15189:2012 certification for quality and competence to run a clinical laboratory , 4. To mOther standards and recommendations include those from the Royal College of Pathologists’ “The retention and storage of pathological records and specimens” . These oCompleting adequate staff training was imperative and staff who built the necessary workflows and resources of the SARS‐CoV‐2 ELISA assay trained other staff to become competent in all processes of the pipeline. Those members of staff trained others until all were fully trained. A training log was kept for each member of staff as part of the audit process and updated when new assays were introduced. The team received information regarding how sample handling adhered to the Human Tissue Act 2004 in the United Kingdom and training in the following areas:Good Clinical Practice (GCP) and the legal framework of a diagnostics lab.Data protection laws (the GDPR and UK Data Protection Act 2018) .Auditability was achieved through recording processes within the pipeline. Internal quality control (QC) standards for the assays must be kept as specified in the ISO 15189:2012 standard. The following reports were stored and made accessible:Instrumentation QC, maintenance history and validation results.Reagents QC including validation batch numbers.External Quality Assurance (EQA) using a national or internationally recognized body, such as NEQAS in the United Kingdom .Maintenance and calibration records for equipment for example, pipettes used in the pipeline, must be kept for up to the lifetime of the equipment plus 4 years .The temperature of fridges and freezers via a sample management system to give an independent secondary recording.Access to sample material was restricted in the interest of safety, information security, and confidentiality. Other governance comes from the Crick’s institutional policy documents.The final step of the pipeline was reporting serology test results to a UKAS accredited medical laboratory by a certified Biomedical Scientist (BMS). The BMS’s responsibility is to oversee the process and to ensure the results meet the specific standards set out above. They are given the processed results of the tests, as well as the raw data, to assess the quality and ensure they meet the standards for a clinical virologist to make a diagnosis.The audit trail formed the basis for the development of the sample reception process. At every decision point in designing the pipeline the primary concern was to ensure the sample could not be separated from its result. A two‐person approach, as outlined in Figure Logs were created to identify the sample status across the entire pipeline and allowed identification of potential breakpoints along the way. For each breakpoint a contingency plan was developed in case of errors as shown in Table As part of this process quarantine measures were introduced ensuring that unscheduled samples that arrived without manifests, or samples with specific rejection criteria were stored in a separate area. This was a location within the fridge that was separated by quarantine criteria notifying the team that the reporting body should be contacted for further instruction before samples were processed. This removed the chance of processing a sample that was not meant to be tested or one that met rejection criteria which would potentially invalidate the result.As the pipeline developed, samples were received from multiple sources for various uses which required either diagnostic, research based or quality control output channels. The sample reception process was evolved to accommodate this.The pipeline was started manually to get it working as quickly as possible and to enable a more efficient and timely digital development process. The parameters for a Laboratory Information Management System (LIMS) evolved from building the pipeline physically from scratch. Pressure tests in the form of dummy runs with batches of mock samples were undertaken to test the robustness of the manual pipeline. These steps were then used as a framework for digitization as shown in Table Once all the parameters had been defined and tested automating the process removed multiple sources of potential human error. This increased not only the efficiency of the pipeline by reducing time spent performing manual steps but also freed up time spent by staff performing the tasks.Initially, we used a commercial electronic sample tracking system already in place at the Crick. After collaborating with the Scientific Computing STP a web app‐based LIMS was developed that encompassed all the specific requirements and parameters the commercial option could not. This custom‐built system relied upon barcoding and robot technology to electronically track the samples through the pipeline. After further rounds of dummy runs and the creation of new SOPs, to include the new LIMS, the pipeline was fully automated leaving the manual system in place as a contingency for any potential point of failure.Despite the abundance of recent studies on SARS‐CoV‐2 rigorous testing and knowledge of the immune response to the disease was still lacking. This posed a problem with reporting an accurate serological evaluation. Other ways of testing for SARS‐CoV‐2‐specific antibodies were developed including both ELISA and flow cytometry assays with cells and beads. The development of such tests for diagnostic purposes required each variable to be tested and validated. There is currently no gold standard for SARS‐CoV‐2 serological tests so we used different commercial assays to cross validate our internal assays. Although mostly concordant, the different tests showed disparity between some positive samples. The disparity may be due to biological differences or varying assay sensitivities. Importantly, the negatives remained concordant.n > 30) were carried out comparing the reproducibility of the robot to human pipetting.Unlike in research, a diagnostic pipeline requires strictly defined protocols to minimize variability and ensure reliable reporting to external parties. The team identified steps in the pipeline where human error could possibly be introduced and incorporated extra checks or contingencies to prevent this. The first obvious step for introduction of human error was in the creation of the 96‐well master plate. As well as human pipetting errors there was potential to introduce mismatches between sample position and sample ID. We therefore automated this step using a robot to decant samples from their tubes into a recorded position on the master plate. As the robot had not been previously used in a diagnostic pipeline several tests and validations had to be done. At least five runs of an ELISA assay with large sample numbers  to measure the variability of samples both within and between each run. We found that dispensing samples using the robot gave an intra‐assay % CV of 13.07 ± 14.86 (mean ± SD) while human pipetting resulted in an intra‐assay % CV of 7.85 ± 4.74%. Conversely, robotic and human pipetting resulted in interassay % CVs of 19.45 ± 7.51 and 23.50 ± 11.63 respectively as shown in Figure n = 45) and negatives (n = 40) as previously confirmed by an independent laboratory through PCR and a commercially available assay. The criterion set for specificity was that tests should not report a negative sample as positive. The criterion for sensitivity was that tests should not report a positive sample as negative. Finally, for reproducibility the criterion was that results should be consistent among the five different repeats. Our results showed the tests were reproducible, specific and sensitive as defined in Table For validation it was necessary to demonstrate specificity, sensitivity and reproducibility within the results of the pipeline , 9. UsinMost diagnostic assays do not include standard curves because they are designed to provide a “YES” or “NO” answer. During the process of pipeline validation however, we included standard curves in some tests. These standard curves provided us with parameters such as limits of detection  and dilution linearity as shown in Figure While our methods covered all the parameters required for confidence it remained necessary to evaluate each sample individually. It was imperative to evaluate sample stability because assays could be conducted at different time points. For this the same set of samples kept at 4°C were tested at different time points over 2 weeks.In June 2020 lockdown restrictions were eased; members of the Crick started to return to work in a phased approach and normal staff duties began to increase. The decision was made to split the Flow STP into two groups on a rota basis to enable social distancing and to prevent the entire team having to self‐isolate if one member tested positive for COVID‐19. Careful management of staff time was required taking into consideration both annual and sick leave requirements.This reduction in staff numbers combined with the running of the clinical diagnostic pipeline had a major impact on the services the Flow STP could provide. The pre‐COVID workload could not be sustained and was streamlined. Prior to the lockdown we had pretrained scientists who were able to perform cell sorting both for themselves and their colleagues. Post lockdown this relieved a large burden on the Flow STP and enabled scientists to continue their research independently. The online booking system was adapted and we implemented a more consultative approach to staff planning and cell sorting which helped triage service requests.The Flow STP continues to aid researchers with COVID‐19 experiments. As previously discussed flow‐based serological assays continue to be under development internally. These will be added to the clinical diagnostic pipeline based on their success. The reality of the “new normal” for the Flow STP at the Crick is to continue and build on our work on serology assays as a clinical diagnostics lab as well as maintaining an effective flow cytometry service to the Crick research labs. Although our time has been repurposed, putting constraints on our usual duties, we are delighted to contribute our newly adapted services to assist in this global pandemic.The challenges faced by the Flow STP can be separated into those created by COVID‐19 regarding the changes required to everyday working practices and those posed by the uncertainty and novelty of creating the pipeline as outlined in Figure Emma Russell: Conceptualization; formal analysis; project administration; validation; writing‐original draft; writing‐review and editing. Ana Agua‐Doce: Conceptualization; formal analysis; project administration; validation; writing‐original draft; writing‐review and editing. Lotte Carr: Conceptualization; formal analysis; project administration; validation; writing‐original draft; writing‐review and editing. Asha Malla: Writing‐original draft; writing‐review and editing. Kerol Bartolovic: Conceptualization; writing‐original draft; writing‐review and editing. Dina Levi: Writing‐original draft; writing‐review and editing. Carl Henderson: Writing‐original draft; writing‐review and editing. Debipriya Das: Writing‐original draft; writing‐review and editing. Hefin Rhys: Conceptualization; formal analysis; project administration; validation; writing‐original draft; writing‐review and editing. Philip Hobson: Conceptualization; formal analysis; project administration; validation; writing‐original draft; writing‐review and editing. Sukhveer Purewal: Conceptualization; project administration; writing‐original draft; writing‐review and editing. Andrew Riddell: Conceptualization; formal analysis; project administration; resources; writing‐original draft; writing‐review and editing.The following rationale was applied to conclude that no ethics approval was required to conduct this assay development:Sera is not HTA relevant material.Samples used were leftover serum samples from routine clinical testing of individuals, not taken for a purpose within the remit of Research Ethics Committees.Individual samples were anonymized such that no personal identifying information was present.These samples were provided with appropriate consents from the source.Appendix1. Documentation required for pipeline development including relevant trainingClick here for additional data file.