Extravascular body fluids (EBF) analysis can provide useful information in the differential diagnosis of conditions that caused their accumulation. Their unique nature and particular requirements accompanying EBF analysis need to be recognized in order to minimize possible negative implications on patient safety. This recommendation was prepared by the members of the Working group for extravascular body fluid samples (WG EBFS). It is designed to address the total testing process and clinical significance of tests used in EBF analysis. The recommendation begins with a chapter addressing validation of methods used in EBF analysis, and continues with specific recommendations for serous fluids analysis. It is organized in sections referring to the preanalytical, analytical and postanalytical phase with specific recommendations presented in boxes. Its main goal is to assist in the attainment of national harmonization of serous fluid analysis and ultimately improve patient safety and healthcare outcomes. This recommendation is intended to all laboratory professionals performing EBF analysis and healthcare professionals involved in EBF collection and processing. Cytological and microbiological evaluations of EBF are beyond the scope of this document. However, the term EBF should be distinguished from the term non-standard body fluids, since not all EBF are non-standard samples. For example, urine and CSF, although extravascular by definition, are considered “standard fluids” when analysing total proteins and/or glucose  analysed in the clinical laboratory comprise cerebrospinal fluid (CSF), serous fluids , synovial fluid, amniotic fluid, drain fluid, semen, urine, dialysate and others. The term “non-standard” body fluids is frequently used in the literature, referring to all body fluids lacking manufacturer’s analytical performance specifications  of the Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM), is to assist in the attainment of national harmonization of EBF analysis and ultimately improve patient safety and healthcare outcomes. The role of laboratory professionals is not only to ensure reliable EBF test results, but also to encourage the appropriate use of EBF analyses and provide guidance in results interpretation. Thus, this recommendation is intended primarily to all laboratory professionals performing EBF analysis, but also to all healthcare professionals involved in EBF collection and processing.Recognizing the importance of harmonization in the field of laboratory medicine, the CSMBLM has so far published several national recommendations  were used as a framework while writing this document . Finally, relevant articles referenced in articles retrieved in the initial search were used.This document is based on results of the Croatian survey on laboratory EBF testing which gave an insight on the current state of procedures used in EBF analysis in Croatia. The main findings of the survey showed that procedures used in EBF laboratory testing across Croatia are not harmonized and deviations from desirable procedures were detected in all phases  of EBF analysis , since these fluid types are analysed in almost all laboratories in Croatia  on automated analysers, which makes it widely available and relatively inexpensive  should be discouraged and discontinued  if available, should provide an insight of the most frequently analysed EBF types and analyses in order to plan appropriate verification/validation procedures. Assays that do not add value to patient management (e.g. available CLSI documents) /reportable range evaluation, and, if applicable, verification of manufacturer supplied reference intervals. Procedures and principles for method verification of “standard fluids” should be applied to EBF verification  , AMR/reportable range, and, if necessary carry-over, clinical sensitivity and clinical specificity. All validation experiments should be performed similarly to “standard fluid” protocols (i.e. in mixing studies). Different approaches might be applied depending on the availability of EBF samples with high/low analyte concentration. For example, if one EBF sample with low and one with high analyte concentration are available, their mixtures with increasing concentrations of the target analyte across the AMR should be produced and a 5-point curve should be generated. Alternatively, a standard sample type, standard solution or calibrator with known high concentration of the target analyte are used for spiking an EBF sample with low analyte concentration. Matrix interference is evaluated by comparing measured results to expected target analyte concentrations. In either case, if the reproducibility is comparable to standard samples and/or dilution ratios are recovered, the existence of matrix effect can be reasonably excluded  mode offer an automated solution for cell counting and differentials in various body fluids see . These s2.Quality control testing for EBF analyses should be instituted in accordance with quality management strategies already present in the laboratory. The QC materials  should be analysed in the same manner as routine EBF samples  programmes are available for EBF analyses, laboratories should participate  of the analyser fluidic system. The limits of such background count should be defined by individual laboratories depending on the EBF type tested  is formed by plasma ultrafiltration in the capillary endothelium of the pleural cavity. Its function is to lubricate the motion of pleural membranes against each other. In case of pleural fluid accumulation, due to imbalance in fluid formation and/or absorption, a pleural effusion is formed , collection date and time, the working diagnosis, hospital ward, identification of the ordering physician and its contact details, identification of the clinical staff that performed collection. Tests requested and any clinically relevant information (e.g. diuretic therapy) to facilitate results interpretation should be clearly indicated on the test request. Additionally, the collection procedure , collection site and anatomic origin of the sample should be clearly stated (Class 1)  or it should be cancelled , location, date and time of collection and anatomic site of collection (Class 1) , with at least two unique identifiers (Sample containers not labelled properly (or unlabelled) should not be accepted for analysis (3.1.1.3(i.e. transport and processing) used for pleural fluid analysis should be dictated by the tests ordered . It should be processed promptly upon receipt.For interpretative purposes, a serum sample should be collected within one hour from pleural fluid collection and sent to the laboratory (Class 1) (e.g. ultrasound-guided). Thoracentesis is performed for either diagnostic or therapeutic purposes (or both). It involves pleural fluid aspiration from the pleural space using a needle. Although not under direct laboratory supervision, the collection technique used might greatly affect EBF analysis results. Thus, clinical practice guidelines should be followed in order to standardize procedures used in thoracentesis  used for thoracentesis, containing pleural fluid sample, are not acceptable containers. Large sample volumes  collected in thoracentesis syringes should be transferred into appropriate containers at the collection site before transportation to the laboratory. This is particularly important in avoiding sample clotting (particularly if the sample is haemolysed) (per serum centrifugation conditions (Tubes containing anticoagulants should be mixed according to manufacturer’s instructions to ensure proper mixing and avoid clot formation (i.e. electrolytes and metabolites). However, due to limited preanalytical data, the feasibility of performing all pleural fluid analyses from pleural (as well as other serous) fluid samples collected in lyophilized syringes is unclear  and transported to the laboratory immediately. Pleural fluid samples for pH determinations should be collected and analysed similar to whole blood samples for blood gas analysis. Contemporary blood gas analysers are able to measure not just blood gas parameters but also related measurements . If limited (or insufficient) sample volumes (less than those acceptable) are submitted for analysis, test priority should be established in consultation with the ordering clinician   should be considered  might be used to broadly indicate pleural fluid pH values in case of suspected extreme pH. The application of urine dipsticks in assessing pleural fluid extreme pH values poorly investigated in the literature  from transudates (caused by systemic disease) (Class 1) . If turbidity remains after centrifugation, it is most likely that a chylous or pseudochylous effusion is present and lipid analysis is warranted  in pleural fluid and serum. Accordingly, an exudative effusion meets at least one the following criteria:pleural fluid/serum protein ratio > 0.5, and/orpleural fluid/serum LD ratio > 0.6; and/orabsolute pleural fluid LD activity > 2/3 of the serum upper reference limit (URL) (Class 1) , serum-pleural fluid albumin gradient, pleural fluid cholesterol, pleural fluid/serum cholesterol ratio, or combinations of pleural fluid cholesterol, pleural fluid LD and pleural fluid protein may help in differentiation (Class 1)  present in the sample   is not analytically challenging as is the case with ascites .Pleural fluid and serum measurements used for calculation of ratios and gradients should be obtained using the same method. Additionally, according to available literature data, the determination of analytes in pleural fluid (i.e. no concomitant blood sample is required)  determination in pleural effusions should be confined to cases with clinically suspected cardiac pleural effusions that meet exudative Light’s criteria, specifically when effusions are bloody or following diuretic therapy. Heart failure (HF) accounts for 80% of transudative pleural effusions. N-terminal pro-brain natriuretic peptide concentrations are increased in patients with HF. A NT-proBNP concentration > 1500 ng/L in pleural fluid achieves 94% sensitivity and 89% specificity for the diagnosis of HF  (Class 1).Total white blood cell (WBC) count should not be determined for the differentiation of pleural transudates and exudates . Conversely, basophils are rarely found in pleural effusions. The clinical significance of both these cell types is largely unknown (etc.) found during morphological assessment of pleural fluid sample  and leukocytes by automated counting) should be addressed by informing the ordering physician in order to suggest the need for cytological evaluation .Atypical cells  should be determined in pleural effusions if pancreatitis, malignancy, oesophageal rupture, pancreatic pseudocyst and liver cirrhosis are suspected (Class 1) . Pancreatic AMY isoenzymes will be higher in pleural effusions related to pancreatic diseases (with high lipase activities). The interpretation of high AMY activities in pleural effusions is presented in Pleural fluid adenosine deaminase (ADA) is an accurate and useful indicator of tuberculous pleurisy. It should be measured in patients with suspected tuberculous effusions to differentiate tuberculous and malignant effusions (Class 2)  strongly suggests a complicated parapneumonic effusion requiring surgical evacuation (Class 2) . If the measured pleural fluid pH is low (< 7.35), arterial pH might be determined to rule out systemic acidosis  are indicative of complicated parapneumonic effusion, but are also found in malignancy, tuberculosis and rheumatoid disease. Due to low specificity and poorer diagnostic performances in comparison to pleural fluid pH, the measurement of glucose in pleural fluid should be interpreted in correlation with clinical symptoms  (Class 1) . The most common causes are malignancy (e.g. Hodgkin’s lymphoma) and trauma. Despite the similar appearance, chylous effusions should be differentiated from pseudochylous effusions, due to their different aetiology and treatment approaches. Since chyle is composed of chylomicrons, high triglyceride concentrations are expected. Conversely, pseudochylous (or chyliform) effusions are cholesterol rich, and are caused by chronic pleural inflammation . Usually, pleural fluid triglycerides and cholesterol are measured simultaneously to exclude pseudochylotorax presence (Chylous effusion (or chylothorax) results from chyle or lymph accumulation in the pleural space due to leakage from the thoracic duct or other lymphatic vessels . A pleural fluid to serum creatinine ratio > 1 is considered a hallmark of urinothorax, but should always be interpreted in relation to other clinical findings (Class 1)  is a rare cause of pleural effusion and may be caused by obstruction due to malignancy, fibrosis or calculus, blunt or surgical trauma. Creatinine is considered a sensitive and specific indicator of urine leakage presence. The urinothorax fluid sample is usually transudative , but can erroneously be classified as exudative if high LD activities are measured in the same sample. This must be taken into account when interpreting Light’s criteria in correlation with high clinical suspicion for urinothorax. However, urinothorax is characterized with markedly elevated creatinine concentrations in the pleural fluid sample (177 - 884 μmol/L) in comparison to creatinine concentrations found in simultaneously collected serum samples. Furthermore, pleural fluid to serum creatinine ratio is considered the biochemical criterion for urinothorax diagnosis: in case of urinothorax its value is > 1  (i.e. 50-60%) in differentiating malignant causes of pleural fluid accumulation. Various tumour markers have been investigated for the purpose of improving differentiation of malignant from non-malignant effusions including carcinoembryonic antigen (CEA), cancer antigen (CA) 19-9, CA 125, cytokeratin 19 fragment (CYFRA 21-1), CA 15-3, neuron specific enolase (NSE) etc. However, their usefulness is limited due to low sensitivity  on the test report and the ordering clinician should be contacted to explain these limitations  , at 4°C and at - 20°C for 7 days. Pleural fluid cholesterol is stable for 4 days at room temperature, and for 14 days at 4°C and - 20°C. Pleural fluid triglycerides are stable for 4 hours at all three temperature conditions indicated. Glucose in pleural fluid samples is stable for 2 hours at room temperature, and 7 days at 4°C and - 20°C. The stability of pleural fluid LD is limited to 4 hours at room temperature and 1 day at 4°C, whereas due to LD instability at - 20°C, pleural fluid samples should not be frozen . The comments should be standardized (predefined) by the laboratory, written in clear and unambiguous language  fill the pericardial cavity and allow the heart to easily move during contraction and relaxation. The most frequent cause of pericardial effusions is acute pericarditis of bacterial, tuberculous or fungal origin. Furthermore, pericardial effusions can be associated with myocardial infarction, malignancy, uraemia or mediastinal injury  .3.2.23.2.2.1Pericardial fluid appearance should be determined upon sample acceptance and before centrifugation. Pericardial fluid appearance should not be used as a definitive test to differentiate transudates from exudates (Class 1)  fluids are produced in infections or malignancies. Bloody pericardial fluid may be caused by cardiac rupture or puncture of a ventricle during pericardiocentesis, or traumatic pericardiocentesis. Milky pericardial fluid appearance suggests the presence of chylopericardium , demonstrated good diagnostic performances . HoweverSimilar to pleural effusions, the highest misclassification rates of transudates as exudates were found in patients receiving diuretics. Albumin gradient ≤ 12 g/L was used to identify pericardial fluid exudates, yielding a sensitivity and specificity of 90% and 89%, respectively. An exudative pericardial fluid cholesterol cut-off point of ≥ 1.6 mmol/L yielded a sensitivity and specificity of 71% and 83%, respectively   and/or the confirmation of a specific diagnosis that caused the effusion .3.3The peritoneal space is a mesothelial lined space which physiologically contains up to 50 mL of peritoneal fluid formed by the ultrafiltration of plasma  studies are procedures that allow detection of small volumes of peritoneal fluid and are helpful in assessing the possible cause of ascites. However, diagnostic paracentesis is considered essential in all patients with ascites prior to therapeutic interventions to exclude spontaneous bacterial peritonitis (SBP) and causes of ascites other than cirrhosis. Furthermore, diagnostic paracentesis is indicated in patients with new-onset ascites, in patients requiring hospitalisation due to ascites and in those with ascites accompanied with unexplained clinical worsening .3.3.23.3.2.1Ascites appearance should be determined upon sample acceptance and before centrifugation. Ascites appearance should be used as an aid in elucidating its aetiology, not as the sole criterion for differential diagnosis of fluid accumulation (Class 1) (i.e. chylous or pseudochylous effusion abundant in chylomicrons with high triglycerides concentrations); purulent ascites is associated with intra-abdominal infection. The possible interpretation of peritoneal fluid appearance is presented in Normally, ascites is clear and pale-yellow. If bloody appearance is caused by traumatic tap, the fluid tends to clot when left standing after collection and eventually clears up. The persistence of milky appearance after centrifugation indicates the presence of lymph , xudative    for peritoneal fluid differentiation , and those related to normal portal pressure  , which might result in incorrectly low SAAG calculation. Spectrophotometric methods for albumin determination overestimate albumin concentrations in the lower concentration range, compared to immunochemical methods . Furthermore, globulin concentrations contribute to oncotic pressure but are inversely correlated to albumin; thus, the presence of hyperglobulinemia (> 50 g/L) might cause falsely low SAAG  might be determined by using spectrophotometric (bromcresol green) methods. Alternatively, nephelome-tric methods might be used. Measuring albumin concentrations in the lower concentration range is analytically challenging. Patients with cirrhosis might exhibit very low serum albumin concentrations  (Class 1).Peritoneal fluid neutrophil count ≥ 250 x106/L), ascitic neutrophil counts should be used when diagnosing SBP. Neutrophil count cut-off values of 250 x106/L and 500 x106/L for SBP diagnosis have similar diagnostic accuracies; however the former cut-off displays better sensitivity, while the later better specificity. If the peritoneal sample is grossly bloody (with a fluid red blood cell count > 10 x109/L), one neutrophil should be subtracted from the neutrophil absolute count every 250 red blood cells to adjust for the presence of blood   fluid in the absence of any other intra-abdominal source of infection. It is a frequent complication in cirrhotic patients with ascites and is caused by translocation of bacteria from the intestine into the peritoneal cavity. Its prevalence in cirrhotic hospitalised patients with ascites is estimated between 10-30%. Although high total WBC in ascitic fluid provides quick information on the presence of infection and is still determined in the diagnosis of SBP irrespective of differential count (diagnostic cut-off > 500 x10i.e. the leukocyte esterase test) have been proposed as rapid and available tool for the early detection of high neutrophil counts in peritoneal fluid. However, urine strips displayed low sensitivity and high false negative results, especially in cases of SBP and low neutrophil count. Therefore, they are not recommended for the rapid diagnosis of SBP. The advances in automated technologies for cell counting and differentials in EBF have prevailed over qualitative methods like urine strips . Ascitic/serum triglycerides ratio > 1 or ascites triglycerides concentrations > 1.2 mmol/L are used for the identification of chylous ascites (Class 1) . Since chylous ascites is usually rich in tryglicerides, the additional measurement of cholesterol in ascites is not necessary. Triglycerides concentrations depend on patient nutritional status, it is imperative that the serum sample is collected simultaneously  are usually associated with pancreatic damage (Class 1)  (Class 2) (e.g. due to cirrhosis). Higher cholesterol concentrations are inherent to malignant ascites due to increased permeability, cholesterol synthesis and cholesterol release form malignant cells. An ascites cholesterol cut-off of > 1.2 mmol/L yielded a diagnostic sensitivity of 93% and diagnostic specificity of 96% for the differentiation of malignant ascites . Low glucose concentrations (e.g. < 2.8 mmol/L) found in peritoneal effusions might indicate its increased consumption in the presence of leukocytes and bacteria . Ascitic creatinine and urea concentrations higher than in a concomitant serum sample (with fluid to serum creatinine ratio > 1) suggest urinary bladder rupture  : total protein > 10 g/L, glucose < 2.8 mmol/L and LD > the URL for serum .


We present a case of a 34‐year‐old male who presented with syncope secondary to a large adenoid cystic carcinoma (ACC) of the distal trachea. A computed tomography and flexible bronchoscopy showed almost complete occlusion of the distal trachea. Resection with curative intent was performed, but resection margins were unfortunately not clear. The patient was subsequently offered adjuvant radiotherapy. Tracheal tumours comprise a small proportion of respiratory tract neoplasm, accounting for only about 2% of airway malignancies. Squamous cell carcinoma is the most common tracheal tumour, followed by ACC. Symptoms are usually attributable to the intraluminal component of the tumour causing an obstruction of the airway, resulting in stridor, dyspnoea, wheezing, haemoptysis, and cough. Syncope as a presenting symptom is exceedingly rare. Tracheal tumours comprise a small proportion of respiratory tract neoplasms, accounting for only about 2% of airway malignancies If the tumour is resectable, surgery is the first modality of choice. Incomplete resection is common ACC has a good prognosis but is known for late local recurrence and metastasis for which long‐term follow up is indicated A 34‐year‐old male without significant medical history presented with a 4‐month history of shortness of breath and a non‐productive cough that worsened in the supine position. He eventually presented to a local district hospital with unexplained syncope.No clear cause for his symptoms was identified, and a computed tomography (CT) pulmonary angiogram (CTPA) was performed to rule out pulmonary embolism. No emboli were present, but the scan demonstrated a large intra‐tracheal tumour with near‐complete obstruction of the distal trachea Fig. A.The syncope was ascribed to likely transient asphyxiation due to intra‐tracheal obstruction, and the patient was referred to our unit. We proceeded to perform an urgent flexible bronchoscopy in the sitting position and confirmed a lobulated, smooth, solid, well‐vascularized mass that almost completely obstructed the distal trachea Fig. B. ConvenThe patient was referred for resection of the tracheal tumour, and resection of the distal trachea at the carina and left main bronchus was performed Fig. C. The paTumour excision margins were unfortunately not clear of tumour infiltration on histology, and a surveillance bronchoscopy confirmed macroscopic recurrence of the primary tumour Fig. . AlthougOur patient is currently undergoing adjuvant external beam radiotherapy, which may be followed by brachytherapy, if required, in a sufficient dose initially or for a later recurrence.ACC is a rare malignant tracheal tumour that is characterized by slow growth. Symptoms are generally directly attributable to tracheal obstruction. Syncope, however, is not a common presenting complaint. A CT of the chest and bronchoscopy are the modalities of choice for pre‐resection evaluation and diagnosis The preferred primary treatment is surgical resection with clear margins and tracheal reconstruction when possible Our patient's endobronchial biopsies showed a myoepithelial‐rich tumour with myxoid stroma and no clear histological features of malignancy, and therefore, pleomorphic adenoma was favoured. However, the resected specimen showed unequivocal invasion and a typical cribriform growth pattern of ACC. This highlights the diagnostic difficulty confronting the pathologist in diagnosing salivary gland‐type neoplasm on small biopsies. Many of the common salivary gland‐type neoplasms can show overlapping light microscopic and immunohistochemical features in limited biopsy material. ACC and pleomorphic adenoma share the combination of epithelial and myoepithelial cell differentiation. C‐KIT (CD117) has been reported to discriminate (positive in most ACCs) but only showed weak focal staining on the endobronchial biopsy, which was interpreted to negative ACC has a 5‐year overall survival rate of 50%, and prognosis is mainly determined by tumour margins and metastasis. If there is incomplete resection, adjuvant radiotherapy has been proven to prolong survival. There is no standard dosage. The unusual location of the tumour makes the approach to radiation therapy challenging. As in all cases, it is governed by the combined requirements of a high dose needed for a tumour of this type and the tolerances of the surrounding normal structures; here, most notably the heart and spinal cord as the other adjacent structures have a relatively high radiation tolerance. Brachytherapy will achieve a higher dose differential between the two but carries the risk of missing tumour cells due to its short effective range.If the tracheal tumour is non‐resectable, radiotherapy is the modality of choice Appropriate written informed consent was obtained for publication of this case report and accompanying images.


The aim of this study was to identify the presence of proinflammatory mediators and neutrophils in the synovial fluid of heifers with ARA, induced by an oligofructose overload.Acute ruminal acidosis (ARA) is a metabolic disease of cattle characterized by an aseptic synovitis. ARA is the result of an increased intake of highly fermentable carbohydrates that frequently occurs in dairy cattle subjected to high production requirements. In human joint diseases such as rheumatoid arthritis and gout, several pro-inflammatory molecules are increased in the synovial fluid, including cytokines, prostaglandin E2, IL-6, IL-1β, ATP, lactate dehydrogenase (LDH), albumin, glucose, matrix metalloproteinase-9 (MMP-9), cellular free DNA, NETs, and serpin B1 were analyzed at 0, 9, and 24 h post treatment.Five heifers were challenged with an oligofructose overload (13 g/kg BW) dissolved in water. As a control, a similar vehicle volume was used in four heifers. Synovial fluid samples were collected from the tarso-crural joint and PGE2, serpin B1 and LDH was detected in the joints when compared to the control group. At 24 h, the synovial fluid was yellowish, viscous, turbid, and contained abundant neutrophils. An increase of DNA-backbone-like traps, histone 3 (H3cit), aggregated neutrophil extracellular traps (aggNETs), and serpin B1 were observed 24 h post treatment. Furthermore, albumins, LDH, ATP, MMP-9, IL-6, and IL-1β were increased after 24 h.At 9 h post oligofructose overload, an increase of IL-1β, IL-6, PGE2, were the earliest proinflammatory parameters that increased in the synovial fluid of animals with ARA. Furthermore, the most sever inflammatory response in the joint was observed after 24 h and could be associated with a massive presence of neutrophils and release of aggNETs.The overall results indicate that IL-1β, IL-6 and PGEThe online version of this article (10.1186/s12917-019-1974-x) contains supplementary material, which is available to authorized users. Acute ruminal acidosis (ARA) is a metabolic-nutritional disease that affects production efficiency, mainly in dairy cattle , 2. A hi2 (PGE2) have been found in the synovial fluid of patients with osteoarthritis  were used. Analysis of membranes was performed by using a LI-COR OdysseyFc system  and densitometry was quantified using the Image 1.51j8 software.Synovial fluid (stored at − 80 °C) was used to quantify total protein using the Bradford colorimetric method . For thi3cit)  was incubated overnight at 4 °C. The following day the secondary antibody, Alexa Fluor 594 , was incubated for 2 h at RT. Finally, DNA was stained with Quant-iT™ PicoGreen™ dye . Images were acquired with an OLYMPUS® Fluoview 1000 confocal microscope.Aliquots of 100 μl of fresh synovial fluid were placed on slides and fixed with 2% paraformaldehyde for 15 min at RT, then washed twice with cold PBS. Primary antibody histone H3 [citrulline R2 + R8 + R17] .Since our data do not show normal distribution and variance homogeneity according to Shappiro-Wilks and Bartlett’s test respectively, a Mann-Withney test for non- parametric data was used. A To experimentally induce ARA in heifers, we used the oligofructose overload protocol . A decreThe synovial fluid of the animals before induction, at 0 h, was colorless, transparent, and had a fluid consistency; similar findings were observed after 9 h. At 24 h, the animals were weak, slow-moving, prone to decubitus, anorexic, and presented explosive liquid diarrhea. At 9 h and 24 h, all animals treated with oligofructose overloads showed lameness resulting in a locomotion score of 2–3 . In addiAs described elsewhere , microbi2, ATP and MMP-9 in the synovial fluid, were assessed in animals with ARA. At 9 h IL-1β, IL-6, PGE2 showed a significant increase compared to the vehicle which was more evident after 24 h  at 24 h  and biochemical  parameters of heifers with ARA. (DOCX 14 kb)


Development of chemo−/radioresistance is a major challenge for the current prostate cancer (CaP) therapy. We have previously demonstrated that epithelial cell adhesion molecule (EpCAM) is associated with CaP growth and therapeutic resistance in vitro, however, the role of EpCAM in CaP in vivo is not fully elucidated. Here, we aimed to investigate how expression of EpCAM is involved in CaP growth and chemo−/radiotherapy response in NOD/SCID mouse models in vivo and to validate its role as a therapeutic target for CaP therapy.EpCAM was knocked down in PC-3 CaP cell line using short hairpin RNA (shRNA). The effect of EpCAM-knockdown (KD) on tumour growth, chemo−/radiotherapy response and animal survival was evaluated on subcutaneous (s.c) and orthotopic mouse models.We found that KD of EpCAM significantly inhibited tumour growth, increased xenograft sensitivity to chemotherapy/radiotherapy, and prolonged the survival of tumour-bearing mice. In addition, we demonstrated that KD of EpCAM is associated with downregulation of the PI3K/Akt/mTOR pathway.In conclusion, our data confirms that CaP growth and chemo−/radioresistance in vivo is associated with over-expression of EpCAM, which serves both a functional biomarker and promising therapeutic target.The online version of this article (10.1186/s12885-018-5010-5) contains supplementary material, which is available to authorized users. Prostate cancer (CaP) is the commonly diagnosed and the second leading cause of cancer-related death in males in the USA in 2017 [RT continues to be one of standard treatment options for the different stages of CaP. Despite recent advances in the approaches of delivering radiation to the prostate, around 30–50% of patients who undergo RT will experience biochemical recurrence within 10 years following treatment , which hDespite the revolutionary targeted therapies and immunotherapy in CaP management, chemotherapy retains a key role in the treatment of advanced CaP disease. Several recent clinical trials showed profound benefits with docetaxel (DTX) by prolonging the survival, at the cost of significant toxicity , 4. DTX . Our results suggest that EpCAM is not only an important functional biomaker in CaP development and therapeutic sensitivity, but also a promising therapeutic target to repress CaP growth and overcome the resistance to chemo−/radiotherapy.Epithelial cell adhesion molecule (EpCAM), also known as CD326, is a transmembrane glycoprotein that is highly expressed in rapidly proliferating carcinomas . Our preDetailed information and conditions for all antibodies are listed in Additional file 2.PC-3 CaP cell line was purchased from American Type Culture Collection  and cultured in RPMI-1640 supplemented with 10% (vol/vol) fetal bovine serum (FBS), 50 U/mL of penicillin, and 50 μg/mL of streptomycin. All cell culture reagents were supplied by Life Technologies Australia Pty Ltd.  unless otherwise stated. The cell line was maintained in a humidified incubator at 37 °C and 5% COThe identity of cell lines was confirmed by short tandem repeat profiling and experiments were carried out within a limited number of passages of initial authentication.Knockdown (KD) of EpCAM were achieved using a previously published method with modification . Three MCells grown on Millicell® EZ slides  were fixed by cold methanol, rinsed by TBS (pH 7.5), blocked in 10% (vol/vol) goat serum, and incubated with rabbit anti-human EpCAM antibody at 4 °C overnight (o/n), and then incubated with Alexa Fluro® 488 goat anti-rabbit IgG (H + L) secondary antibody for 1 h at room temperature (r/t). Negative controls were treated identically, but using non-specific rabbit IgG. Propidium iodide (PI) was used for nuclei staining.Immunofluorescence staining was then visualised using a ZEISS Axio Vert.A1 microscope .Western blot analysis was performed as previously described . BrieflyTotal RNA was isolated with High Pure RNA isolation kit  and 2 μg of total RNA was used to synthesise cDNA using the SuperScript III First-strand Synthesis System Kit , according to the manufacturers’ protocol. qRT-PCR was carried out on a Rotor-Gene instrument  in a solution containing SYBR® Select Master Mix , primer and cDNA in a final volume of 20 μL. GAPDH was used as a reference control.All animal care and experimentation procedures followed the guideline according to protocols approved by the Animal Care and Ethics Committee (ACEC 14/46A) of UNSW Sydney and conformed to the Animal Research Act 1985 and Australian code for the care and use of animals for scientific purposes 8th edition (2013).Male, 6–8 weeks old NOD/SCID mice of a weight of 15-20 g  were housed in a group of 3 or 4, in specific pathogen-free facilities and all experiments were performed in a laminar flow cabinet in the morning of the day unless stated otherwise. House and husbandry conditions were a 12:12 light:dark cycle, mouse maintenance food and water. Environmental enrichment included bedding, one red tinted guinea pig hut and one handful of paper wool.Mice were kept at least 1 week before experimental manipulation. All mice were monitored daily for health status and weight were recorded at least twice a week. All mice remained healthy and active during the experiments.6 PC-3-EpCAM-KD or PC-3-EpCAM-scr CaP cells in 100 μL Dulbecco’s phosphate-buffered saline (DPBS) were injected subcutaneously in the right rear flank region of mice, respectively. Based on our previous study and a Power calculation, tumour progression of 10 mice in each cohort (n = 10) was measured weekly using callipers, and tumour volumes were calculated as 0.5 × (length × width2) (in millimetres) for up to 8 weeks. Upon sacrifice, tumour xenografts were harvested for histological examination and immunohistochemistry (IHC).To establish s.c. xenograft model, as described previously , 10, mic6 PC-3-EpCAM-KD or PC-3-EpCAM-scr CaP cells suspended in 50 μL DPBS were injected into the prostatic lobe after exposure through a lower midline laparotomy incision. Starting from the second week after cell inoculation, based on our previous study and a Power calculation, tumour progression of mice in each cohort (n = 10) was monitored weekly by 3D ultrasound as described in the previous publication [To establish orthotopic xenograft model, mice were randomised into two groups, and 1 × 10lication . Upon saThe dose-tolerance relationship was examined in 6 NOD/SCID mice/per group without tumours following a single i.p. administration of DTX, or saline as VC treatment respectively to determine the maximum tolerance dose (MTD). The candidate doses of DTX were 10, 20, 30, 40, 50 mg/kg, based on our previous publication . The MTD3 in s.c. model in each subgroup, 4 mice/per group (n = 4) including PC-3-EpCAM-KD and PC-3-EpCAM-scr cell lines were treated with 50 mg/kg DTX (MTD) once by i.p. injection or with VC , respectively. Drugs and vehicle administration was randomised. Tumour growth was plotted by measurements using callipers as previously published [When average tumour size reached 70 ± 10 mmublished .3 in orthotopic model in each subgroup, 4 mice/per group (n = 4) including PC-3-EpCAM-KD and PC-3-EpCAM-scr cell lines were treated with 50 mg/kg DTX once by i.p. injection or with VC , respectively, based on the pilot study. Drugs and vehicle administration was randomised. Tumour growth was monitored by ultrasonography and calculated using Vevo® Lab 1.7.0 software  according to our previously published method [When average tumour size reached 50 ± 10 mmd method .The tolerance study was also performed to determine the MTD in mice for RT. The dose-tolerance relationship was examined in NOD/SCID mice without tumours for local radiation treatment. Mice were anaesthetised with 50–80 mg/kg ketamine and 3–5 mg/kg xylazine (i.p.) and immobilised in a custom-designed chamber with a lead shield covering the upper half of their bodies. Mice were irradiated in the X-RAD 320 Biological Irradiator  with only lower abdomen areas being exposed. Two mice were used in each group. Mice in each group received a dose of 2Gy/day for 2, 3, 4 and 5 consecutive days , respectively. A sham control (SC) radiation group was also included. The MTD for RT chosen for subsequent studies was the highest dose without the exhibition of endpoints that were defined in the animal monitoring criteria.3 in s.c. model in each subgroup, 4 mice/per group (n = 4) including PC-3-EpCAM-KD and PC-3-EpCAM-scr cell lines were irradiated at a dose of 2 Gy for 4 consecutive days  or while 4 control mice/per group (n = 4) went through the same procedure with the SC irradiation. Irradiation and sham irradiation exposure was randomised.When average tumour size reached 70 ± 10 mm3 in orthotopic model in each subgroup, 4 mice/per group (n = 4) including PC-3-EpCAM-KD and PC-3-EpCAM-scr cell lines were irradiated at a dose of 2 Gy for 4 consecutive days  or with SC (no irradiation). Irradiation and sham irradiation exposure was randomised.When average tumour size reached 50 ± 10 mm3, 6 mice/per group (n = 6) received no treatment (used as control), DTX treatment , DTX-VC treatment  radiation  or RT-SC treatment (no radiation), respectively. Allocation of mice into each cohort was randomised.NOD/SCID mouse s.c. xenograft model was established using PC-3-EpCAM-KD or PC-3-EpCAM-scr CaP cells as described above. When tumour size reached 70 ± 10 mmTumour size was documented weekly using a calliper for up to 13 weeks. Endpoints were defined in the animal monitoring criteria. Comparison of the survival of mice was made using a Kaplan-Meier curve.Standard immunohistochemistry procedures were used to visualise biomarkers of interest as previously published . BrieflyStaining intensity (0–3) was assessed using a light microscopy . The assessment criteria were as previously reported , where: P values were 2-sided. P < 0.05 was considered significant. The Kaplan-Meier method and logrank test were used for endpoint survival analysis. All statistical analyses were performed using the GraphPad Prism 7.02 software .All numerical data were expressed as mean ± standard deviation (SD). Data from two different groups were analysed using two-tail Student’s t-test. All As shown in Fig. n = 10; P < 0.05), in both s.c. and orthotopic models, respectively.As shown in the tumour growth graphs in Fig. n = 10) became palpable from the third week after cell inoculation in PC-3-EpCAM-KD group while all xenograft tumours (n = 10) became palpable from the second week after cell inoculation in PC-3-EpCAM-scr group. Significant tumour growth difference was found 3 weeks post cell inoculation . After five weeks post cell inoculation, in PC-3-EpCAM-KD group, tumour volume was 106.4 ± 9.5 mm3, while in PC-3-EpCAM-scr group, tumour volume was 183.6 ± 18.4 mm3 . At the end of experiment (7 weeks post cell inoculation), mouse tumours were harvested, measured and weighed. In PC-3-EpCAM-KD group, tumour volume was 222.3 ± 31.1 mm3, tumour weight was 0.5 ± 0.2 g; whereas in PC-3-EpCAM-scr group, tumour volume was 455.8 ± 44.5 mm3, tumour weight was 1.6 ± 0.1 g , suggesting that a significant difference exists in tumour volume and weight between the two sublines. Typical images for tumour sizes of different groups of mice in s.c model at the end of experiment are shown in Fig. In s.c. model, all xenograft tumours . After five weeks post cell inoculation, in PC-3-EpCAM-KD group, tumour volume was 90.8 ± 11.3 mm3, while in PC-3-EpCAM-scr group, tumour volume was 129.7 ± 17.6 mm3 . At the end of experiment (7 weeks post cell inoculation), tumours were harvested, measured and weighed. In PC-3-EpCAM-KD group, tumour volume was 187.2 ± 24.9 mm3, tumour weight was 0.6 ± 0.1 g; whereas in PC-3-EpCAM-scr group, tumour volume was 304.1 ± 24.2 mm3, tumour weight was 1.2 ± 0.2 g , indicating a significant difference in tumour volume and weight in orthotopic model from the two sublines. Typical images for tumour sizes of different groups of mice in orthotopic model at the end of experiment are shown in Fig. In orthotopic model, 3D ultrasound imaging was used to visualise and monitor tumour growth. The prostatic tumour became visible from the second week post cell inoculation in both PC-3-EpCAM-KD and PC-3-EpCAM-scr group. Significant tumour growth difference was found 3 weeks post cell inoculation , PC-3-EpCAM-scr-DTX and PC-3-EpCAM-scr-VC groups (P < 0.01), PC-3-EpCAM-KD-VC and PC-3-EpCAM-scr-VC groups (P < 0.05), respectively. The ratios of DTX-treated versus VC-treated tumour volumes (DTX/VC) were calculated and plotted , suggesting that the KD of EpCAM can increase the DTX sensitivity of CaP tumours in s.c. mouse model. The representative images for tumour sizes at the end of experiment are shown in Fig. In s.c. mouse model, DTX or VC treatment were administered at 4 weeks post cell inoculation. Tumour volumes were plotted against elapse time Fig. . Signifited Fig. . NotablyP < 0.01), PC-3-EpCAM-scr-DTX and PC-3-EpCAM-scr-VC groups (P < 0.01), PC-3-EpCAM-KD-VC and PC-3-EpCAM-scr-VC groups (P < 0.05), respectively. The ratios of DTX versus VC tumour volumes (DTX/VC) were calculated and plotted , suggesting that the KD of EpCAM can increase the chemosensitivity of CaP tumours in orthotopic mouse model. The representative images of tumour sizes at the end of experiment are shown in Fig. In orthotopic mouse model, DTX or VC treatment was given 4 weeks post cell inoculation. Tumour volumes were plotted against elapse time Fig. . Signifited Fig. . OverallP < 0.01), and between the scr groups: PC-3-EpCAM-scr-RT and PC-3-EpCAM-scr-SC groups (P < 0.01), respectively. Additionally, there was a significant difference between SC treatment in KD vs scr: PC-3-EpCAM-KD-SC and PC-3-EpCAM-scr-SC groups (P < 0.05). The ratios of RT versus SC tumour volumes (RT/SC) were calculated and plotted , suggesting that the KD of EpCAM can increase the radiosensitivity of CaP tumours in s.c. mouse model. The representative images for tumour sizes at the end of experiment are shown in Fig. In s.c. mouse model, RT or SC treatment was given 4 weeks post cell inoculation. Tumour volumes were plotted against elapsed time Fig. . Signifited Fig. . Unlike P < 0.01), PC-3-EpCAM-scr-RT and PC-3-EpCAM-scr-SC groups (P < 0.05), as well as between SC treatment in KD and scr: PC-3-EpCAM-KD-SC and PC-3-EpCAM-scr-SC groups (P < 0.05), respectively. The ratios of RT versus SC tumour volumes (RT/SC) were calculated and plotted , suggesting that the KD of EpCAM can also increase the radiosensitivity of CaP tumours in orthotopic mouse model. The typical images of tumour sizes at the end of experiment are shown in Fig. In orthotopic mouse model, RT or SC treatment was given 4 weeks post cell inoculation. Tumour volumes were plotted against elapse time Fig. . Here alted Fig. . Startinp = 0.0004)    of tumour-bearing mice (PC-3-EpCAM-KD) by 21.5 days compared with the control group mice (PC-3-EpCAM-scr s.c. xenografts)  Fig. . Additio07) Fig. , and RT 44) Fig.  when comAs the PI3K/Akt/mTOR signalling pathway plays a very important role in cancer therapeutic resistance including CaP , 15, we To further investigate whether KD of EpCAM in CaP cells affects proliferative potential in CaP, tumour sections from s.c. xenografts of NOD/SCID mice were assessed for proliferation using Ki-67 expression. At the end of the experiments, strong expression of Ki-67 was found in PC-3-EpCAM-scr xenograft while a weak to moderate expression of Ki-67 was observed in PC-3-EpCAM-KD xenograft  and decreased CD31 expression found in the tumour xenografts of PC-3-EpCAM-KD CaP cells compared to PC-3-EpCAM-scr group  was associated with KD of EpCAM. Ki-67 is a human nuclear protein that is present during all active phases of the cell cycle but absent from resting cells, making it an excellent marker for measuring the proliferation of cells in human tumours . Since e+ chemoresistant leukemia subpopulations after chemotherapy in a mouse model, suggesting EpCAM is a promising novel target for the treatment of leukemia [2 versus 50 mg/kg) [DTX is now the most widely used and first line chemotherapeutic agent for CRPC . In our leukemia . Interesleukemia , which o0 mg/kg) , route oOver-expression of EpCAM is also related to cancer radiosensitivity. It was demonstrated that higher incidence of intense expression of EpCAM was found in head and neck squamous cell carcinoma (HNSCC) from the hypopharynx, and patients with strong EpCAM expression were associated with local recurrence after primary RT . Kaori eAlthough multiple previous studies focused on the relationship between EpCAM and survival with patients of CaP, a consensus has not yet been reached –31. In tActivation of the PI3K/Akt/mTOR pathway has been shown to be actively involved in CaP progression , 32, 33 Recent data have demonstrated a pleiotropic role of EpCAM in cancer that is not only limited to regulation of cell-cell adhesion, cell proliferation, migration and signalling , but alsBased on the close association between EpCAM and CaP development and therapeutic responses, it will be of great clinical significance to use EpCAM in targeted therapy alone or in combination with other conventional therapies in the treatment of CRPC. Possible targeted therapies include antibodies to inactivate EpCAM proteins , 45, toxOur data have demonstrated that EpCAM is involved in CaP development, chemo−/radioresistance and prognosis and associated with activation of the PI3K/Akt/mTOR signalling pathway. The findings suggest EpCAM is a promising therapeutic target for overcoming CaP chemo−/radioresistance.Additional file 1:Table S1. List of antibodies used in Western Blot and immunohistochemistry. (DOCX 14 kb)Additional file 2:Table S2. Survival information of the subcutaneous mouse CaP model. (DOCX 13 kb)Additional file 3:Table S3. The staining intensity of various markers in subcutaneous xenografts CaP model. (DOCX 13 kb)


Dipodomys) when patches were already occupied by an existing rodent community relative to when patches were empty. Recovery of kangaroo rat populations was slow on plots with an established community, taking approximately 2 years, in contrast with rapid recovery on empty plots with no established residents (approx. three months). These results demonstrate that the presence of an established alternate community inhibits recolonization by new species, even those that should be dominant in the community. This has important implications for understanding how biodiversity may change in the future, and what processes may slow or prevent this change.Human activities alter processes that control local biodiversity, causing changes in the abundance and identity of species in ecosystems. However, restoring biodiversity to a previous state is rarely as simple as reintroducing lost species or restoring processes to their pre-disturbance state. Theory suggests that established species can impede shifts in species composition via a variety of mechanisms, including direct interference, pre-empting resources or habitat alteration. These mechanisms can create transitory dynamics that delay convergence to an expected end state. We use an experimental manipulation of a desert rodent community to examine differences in recolonization dynamics of a dominant competitor (kangaroo rats of the genus  Ecological theory focused on stable states provides important insights into the conditions that allow communities to shift from one stable state to another . NevertSpecies interactions, often in combination with dispersal or environmental change, can play an important role in determining whether biodiversity shifts from one state to another as well as the dynamics of any resulting transient phase –18. SpecDipodomys), to consisting of only a few transient individuals . Many of our rodent species are territorial and sequester resources in caches, providing exactly the scenario where inferior competitors may be expected to delay colonization of a dominant species. Additionally, because kangaroo rats are important granivores in this system, their removal affects the annual plant community that serves as their resource base [We examined the role of species interactions in impeding biodiversity change using a replicated long-term experiment that manipulated the dispersal of seed-eating rodents into experimental plots . The thrrce base ,23. In 22.2 quadrats placed at permanently marked locations.The experiment was conducted at the Portal Project, a 20 ha study site, 6 km northeast of the town Portal, Arizona located on unceded land of the Chiricahua Apache in the Chihuahuan Desert . Twenty-https://github.com/emchristensen/PlotSwitch) and archived on Zenodo [While data collection began at this site in 1977, this analysis focuses on the time period 2013–2018. In March 2015, we changed the treatments on 12 of the 24 plots. The pre-existing treatments on those 12 altered plots had been in place continuously since 1989. We focus here on three specific treatments: (i) kangaroo rat+ plots: kangaroo rat removal plots that had gates enlarged to become controls (three plots), which allowed the addition of kangaroo rats to pre-existing rodent communities; (ii) rodent+ plots: rodent removal plots that had new gates created to become controls (three plots), which allowed the addition of all rodent species to those plots; and (iii) controls: plots that were control plots for the entire period 2013–2018 (four plots), which serve as our reference plots for assessing the dynamics of our newly created control plots. All data and code for these analyses are available on GitHub (n Zenodo .(a)We used GAMs to assess the effect of treatment on various rodent community metrics over time, using the R package mgcv v. 1.8–23  for R 3.Dipodomys merriami, Dipodomys ordii and Dipodomys spectabilis) on each plot over time. Because we expect inferior competitors to be displaced by the invasion of kangaroo rats, we also constructed time series of pooled numbers of individuals of non-kangaroo rat seed-eating species . Because these two time series consist of count data, we used a Poisson distribution in both GAMs. We found that there were some small but significant differences between plots within treatments, and so included plot-specific smooths in the GAMs as well.To assess the ability of competitively dominant kangaroo rats to colonize suitable patches, we constructed time series of pooled number of individuals of the three species of kangaroo rats found at the site (E ∝ m(3/4) where E is the metabolic rate (or energy) and m is the mass of the individual [To examine effects at the community level, we analysed time series of community metabolic flux of the seed-eating rodent community on each plot. Total metabolic flux represents an estimate of community size and resource uptake of the community as a whole  and is gdividual . We esti(b)Differences in plant community composition were assessed using a partial canonical correspondence analysis (pCCA) and permutational significance tests, controlling for between-year effects . We squ(c)ark  of kangaroo rat species on each experimental treatment type and probability of transition (Psi) between treatment types. We used Akaike information criterion to select the best model from candidate models with or without effect of strata  on S and Psi. Analysis was performed on D. merriami and D. ordii; there were too few captured individuals of D. spectabilis for modelling.To better understand the observed differences in population size of kangaroo rats between treatments, we performed population-level analyses on these species during the period after the treatment change. We examined the rate that new kangaroo rat individuals entered each plot type, and performed a multistrata population model using the package RM. 2.2.6; ), a packsoftware  to estim3.a). Differences between the treatment-specific smooths from the GAM (b) showed that kangaroo rat abundances on rodent+ plots converged to control levels within three months . While kangaroo rat populations on rodent+ plots converged with controls quickly, they also continued to experience large population oscillations that sometimes caused abundances to exceed those on long-term controls for brief periods of time, even after the treatment types converged on average. This is consistent with theoretical work demonstrating that transient dynamics sometimes amplify the initial perturbation, moving the system farther from the equilibrium point before converging to it [b). Unlike rodent+ plots, kangaroo rat populations on the kangaroo rat+ plots appeared less oscillatory and never exceeded abundances on control plots (b). Thus, the treatment types differ in their transient dynamics during the colonization and population increase in kangaroo rats. Because all treatments are embedded in the same habitat matrix where kangaroo rats are an abundant species . This high influx of new individuals lasted 2 years despite the nearly immediate convergence with control plots in terms of overall kangaroo rat abundances, and was driven primarily by D. merriami, the more abundant kangaroo rat species at the site. While new individuals flooded into rodent+ plots, kangaroo rat+ plots exhibited slightly suppressed numbers of new arrivals compared to control plots, despite the fact that these plots—like the rodent+ plots—also initially lacked kangaroo rats. Transition probabilities suggest that the suppressed influx of D. merriami individuals was in part owing to immigration. Dipodomys merriami individuals captured on rodent+ or control plots were much less likely to move to a kangaroo rat+ plot than to another treatment . While the transition probabilities and numbers of new individuals suggest that some mechanism reduced the influx of D. merriami individuals onto kangaroo rat+ plots, once a D. merriami individual settled on a kangaroo rat+ plot, it experienced a higher survivorship and extremely low probability of moving to another treatment type . By contrast, D. ordii exhibited little difference in survival on rodent+ versus kangaroo rat+ plots, though its survival estimate was lower on those treatments than on controls . This complicated picture suggests that the difference in transient dynamics between treatments is owing to some condition on kangaroo rat+ plots that slightly suppressed the influx of new individuals, potentially delaying recovery on kangaroo rat+ plots, despite the fact that conditions on kangaroo rat+ plots did not negatively impact survival or generate increased movement to another treatment type.Differences between treatments in the influx  and efflux  of individuals drove the observed contrasts in transient dynamics on rodent+ and kangaroo rat+ plots in complicated ways, which differ based on which kangaroo rat species is examined. When rodent removal plots were converted to controls, they received substantively higher numbers of new kangaroo rat individuals on average than either long-term control plots or kangaroo rat+ plots  and much lower than control levels on rodent removals  . After all plots were converted to controls, non-kangaroo rat abundances on both the rodent+ and kangaroo rat+ plots quickly converged to control levels within a few months (b). The rapid decline in non-kangaroo rat species is consistent with previous research showing that kangaroo rats are behaviourally dominant over the other, typically smaller, seed-eating rodents [Because plots at the site experience the same weather events and do not differ in their distance from source populations, it seems logical that the difference in kangaroo rat transient dynamics is related to whether or not a plot had a pre-existing rodent community at the time the plot was converted to a control. A pre-existing rodent community could potentially impact the colonization dynamics of kangaroo rats either directly through competitive interactions  or indirectly through impacts on seed availability or the plant community. Examination of the dynamics of the non-kangaroo rat species  suggests that competitive interactions are an unlikely explanation for the differences in transient dynamics. Before the change in treatments in 2015, abundances of the non-kangaroo rat rodents on kangaroo rat removals were higher than on controls plots  and the summer plant community (pCCA permutation test: R2 = 0.04 and p = 0.004). In both cases, however, the proportion of variance explained by treatment was small (less than 5%). Thus, while the experimental manipulations did impact plant composition, these impacts were only weakly different between the rodent removals and kangaroo rat removals during this time period  ensured that only dispersal (not population growth) was required for kangaroo rats to establish control-level populations on experimental plots. Despite these advantages, we observed transient dynamics in kangaroo rat recolonization of experimental plots. The most likely mechanism for this delay is that the previously established species displayed interference competition by sequestering resources in caches, with potentially some role of vegetation change, making these plots less appealing to kangaroo rat immigrants for approximately 2 years. In our case, transient dynamics eventually led to restoration of the control state; however, prolonged periods of transient dynamics provide opportunity for unexpected and complex dynamics, especially in the presence of stochasticity, a common feature of ecological systems ,40.Many common methods of managing wildlife populations  will tend to produce transient dynamics . In our


To simulate different diameters of endotracheal tubes and to verify the fluid dynamics aspects by means of flow and resistance measurements. Fluid dynamics software was used to calculate mean flow and airway resistance in endotracheal tube with a diameter of 6.0, 7.0, 7.5, 8.0, 9.0 and 10.0mm at normal body temperature and under constant pressure. The same measurements were taken in the fusion of the first 22cm of a 9.0mm endotracheal tube with 10.0mm diameter, and with the end part in 12cm of a 6.0mm endotracheal tube with 7.0mm diameter. The fusion of the first 22cm of an endotracheal tube of 10.0mm diameter with the terminal part in 12cm of an endotracheal tube of 6.0mm diameter, preserving the total length of 34cm, generated average flow and airway resistance similar to that of a conventional 7.5mm endotracheal tube. This simulation study demonstrates that a single-sized endotracheal tube may facilitate endotracheal intubation without causing increased airway resistance. This model gained popularity since it proposed easy introduction of the device, and decrease by approximately 50% of flow resistance as compared to conventional cylindrical ETT, with direct impact on reduced respiratory work.7The idea of a modified ETT first appeared in the literature in 1945, when Cole8 It is applied to rigid tubes with constant radius, and expressed by the following formula:A single ETT for adults should, above all, preserve the ideal ventilatory conditions for optimal mechanical ventilation, avoiding increased airway resistance (AWR). This characteristic could be based on Poiseuille’s law, which states that the flow (F) through a given tube results from the difference in pressure (P) from one end to the other, in tube length (l) and radius (r), and fluid viscosity (μ).9The flow is directly proportional to the radius to the fourth power, therefore the tube diameter represents the most important factor determining the flow velocity of a fluid passing through it.To verify the aspects of fluid dynamics considering flow and resistance measurements in different diameters of conventional and fused endotracheal tubes.Laboratório Nacional de Luz Síncrotron , in the city of Campinas , in the Engineering Division in order to calculate the mean flow in ETT of different diameters.The Computational Fluid Dynamics (CFD) software was used in the 2O pressure, at body temperature of 36°C in ETT with total length of 34cm. The mean flow and calculated airway resistance values (cAWR) were recorded in two data points:The simulations were conducted using a constant 25cmH- Data point 1 − conventional ETT (cETT): the flow velocity at the tube entrance and exit, and cAWR were measured in cETT with diameters of 6.0, 7.0, 7.5, 8.0, 9.0, and 10.0cm   .- Data point 2 – Fused ETT (fETT): using the CFD, we developed four types of ETT, which were called fETT. The tubes were fused with 22 plus 12 cm, totaling up 34cm of cETT. The 9.0mm and 10.0mm tubes were 22cm long, and the 6.0mm and 7.0mm tubes were 12cm long, generating the fETT: 9.0/6.0mm; 10.0/6.0mm; 9.0/7.0mm; 10.0/7.0mm   .The flow velocities at the entrance and exit, and cAWR in fETT were measured. In both data points, the value of the entrance and exit flow velocities for each ETT was added and divided by two in order to obtain the mean flow.This is a simulation study with the measurements of fluid dynamic of an ETT model calculated by a software. This research was not conducted with human beings and/or animals, therefore it was exempt of submission to the Internal Review Board (Resolution CNS 466/12).2O/L/s – similar to those obtained with the 7.5mm cETT.For the mean flow and cAWR results, the ANSYS software was used; they are depicted on 10Invasive mechanical ventilation is often necessary for successful treatment of acute respiratory failure. It is, therefore, considered as an important measure capable of saving the lives of critically ill patients.The development of a single-size ETT for the adult population, with variable diameter along the length, seems to be an alternative that does not increase airflow resistance, facilitates OTI especially in the emergency setting, and reduces the need for a wide repertoire of device sizes available to physicians.5 when endotracheal anesthesia in infants and young children was still rare. Endotracheal anesthesia refers to the administration of anesthetic gases through an ETT inserted through the oropharynx or nasopharynx, until it reaches the larynx and trachea. Infant ETT must have a narrow diameter to access the larynx. The narrower tube diameters cause greater airflow resistance and can increase respiratory work. Cole created his ETT to be narrower only where it should be positioned: below the larynx.11 Considering Cole’s hypothesis for adult patients, tubes with smaller diameter would also cause greater resistance during ventilation.Cole tracheal tube was developed by the anesthesiologist Frank Cole in 1945,12 If developed for adult patients, such models could generate benefits as greater chance of success when inserting an ETT. In addition, a possible reduction in expenditure could be considered.All prototype ETT designed to date focus on the pediatric population and are still rarely used.2O/L/s) similar to a 9.0/7.0 fETT (mean flow (127.7Lpm), and cAWR (11.74cmH2O/L/s). However, despite the similar values, the use of the 9.0/7.0mm fETT might not be as favorable as the 10.0/6.0mm fETT as to the ease of intubating the patient with a smaller distal diameter tube.The results found in this study demonstrated that the 8.0mm cETT presented mean flow (127.0Lpm) and cAWR  permite não somente uma ventilação eficiente, mas também evita a possível entrada de conteúdo gástrico e oral para o interior dos pulmões, quando dotado de balonete ou cuff .3A intubação orotraqueal (IOT), procedimento utilizado em casos em que há necessidade de ventilação mecânica invasiva, foi descrita pela primeira vez em 1543 por Andreas Vesalius.6 propôs um modelo para ser utilizado em neonatologia e pediatria. Esse modelo ganhou popularidade devido à proposta de fácil introdução do dispositivo e redução de aproximadamente 50% da resistência ao fluxo em relação ao TET cilíndrico convencional, com impacto direto na diminuição do trabalho respiratório.7A ideia de um TET modificado já data na literatura desde 1945, quando Cole8 É aplicada a tubos rígidos, raios constantes e expressa pela seguinte fórmula:Um TET único para adultos deve, sobretudo, preservar a condição ventilatória ideal para a ventilação mecânica ótima, evitando aumento da resistência das vias aéreas (Rva). Essa característica pode ser baseada na lei de Poiseuille, que estabelece que o fluxo (F), por meio de um determinado tubo, é resultado da diferença de pressão (P) de uma extremidade a outra, do comprimento (l) e raio (r) desse tubo, além da viscosidade (µ) do fluido.9O fluxo é diretamente proporcional à quarta potência do raio, o que demonstra que o diâmetro do tubo representa o papel principal dentre todos os fatores determinativos da velocidade do fluxo de certo fluido por meio deste.Verificar os aspectos fluidinâmicos considerando medições de fluxo e resistência em diferentes diâmetros de tubos endotraqueais convencionais e fundidos.Computational Fluid Dynamics (CFD), no Laboratório Nacional de Luz Síncrotron em Campinas (SP) Brasil, na Divisão de Engenharia para calcular o fluxo médio em diferentes diâmetros de TET.Foi utilizado o software 2O, temperatura corporal de 36°C em TETs com comprimento total de 34cm. Os valores de fluxo médio e resistência calculada de vias aéreas (Rcva) foram registrados em dois momentos:As simulações foram realizadas utilizando pressão constante de 25cmH- Momento 1 − TET convencional (TETc): foram medidos os valores da velocidade de entrada e saída de fluxo e Rcva em TETc com diâmetros de 6,0, 7,0, 7,5, 8,0, 9,0 e 10,0cm   .- Momento 2 − TET fundido (TETf): desenvolvemos, pelo CFD, quatro tipos de TET, que denominamos de TETf. Os tubos foram fundidos com 22 mais 12cm, totalizando o tamanho de 34cm do TETc. Os diâmetros de 9,0 e 10,0mm tinham 22cm, e os diâmetros de 6,0 e 7,0mm tinham 12cm de comprimento, criando os TETf: 9,0/6,0mm; 10,0/6,0mm; 9,0/7,0mm; 10,0/7,0mm   .Foram medidos os valores da velocidade de entrada e saída de fluxo e Rcva em TETf. Em ambos os momentos, o valor do fluxo de entrada e saída de cada TET foi somado e dividido por dois, para obter o fluxo médio., envolvendo medidas de fluidodinâmica de um modelo de TET, calculadas por um software. Por não se tratar de pesquisa envolvendo seres humanos e/ou animais, o presente estudo dispensou submissão à apreciação da Comissão de Ética em Pesquisa (Resolução CNS 466/12).Trata-se de estudo de simulação 2O/L/s − valores semelhantes aos de um TETc de 7,5mm.Os resultados de fluxo médio e Rcva foram obtidos utilizando o software da ANSYS e estão descritos na 10A ventilação mecânica invasiva é frequentemente necessária para o sucesso do tratamento da insuficiência respiratória aguda. Assim, considera-se como medida importante e capaz de salvar a vida dos pacientes em estado crítico.O desenvolvimento de um TET de tamanho único para a população adulta, com diâmetro variável ao longo do comprimento, parece ser alternativa que não aumenta a resistência ao fluxo de ar, facilita a IOT especialmente em cenários de urgência e emergência, e reduz a necessidade de amplo repertório de tamanhos de dispositivos à disposição do médico.5 quando a anestesia endotraqueal em lactentes e crianças pequenas ainda era incomum. Anestesia endotraqueal refere-se à administração de gases anestésicos por meio de um TET inserido através da orofaringe ou nasofaringe, até alcançar a laringe e traqueia. Os TET infantis precisam ter diâmetro estreito para acessar a laringe. Os diâmetros dos tubos mais estreitos causam mais resistência ao fluxo de ar através do tubo e podem aumentar o trabalho respiratório. Cole criou seu TET para ser mais estreito apenas onde deveria estar posicionado: abaixo da laringe.11 Considerando a mesma hipótese de Cole, para pacientes adultos, tubos com menor diâmetro também causam maior resistência durante a ventilação.O tubo traqueal de Cole foi desenvolvido pelo anestesiologista Frank Cole, em 1945,12 Se desenvolvidos para os pacientes adultos, tais modelos poderiam gerar benefícios como maior chance de sucesso ao inserir o TET. Além disso, uma possível redução nas despesas poderia ser avaliada.Todos os protótipos dos TETs projetados até o momento se concentram na população pediátrica e ainda são raramente utilizados.2O/L/s) semelhantes a um TETf 9,0/7,0 em relação ao fluxo médio  e Rcva . No entanto, apesar dos valores serem próximos, a utilização do TETf 9,0/7,0mm poderia não ser tão benéfica em comparação ao TETf 10,0/6,0mm em relação à facilidade de intubar o paciente com um tubo que possui diâmetro distal menor.Os resultados encontrados neste estudo demonstraram que o TETc de 8,0mm apresentou fluxo médio  e Rcva (11,81cmHcuff. Porém, neste estudo de simulação, não foram desenvolvidos TET com cuff , e outros estudos são necessários para avaliar a necessidade ou não do balonete.A ideia de utilizar um TETf com diâmetro distal de 6,0mm é facilitar a intubação em cenários de urgência e emergência, e que também possa ser utilizado nas intubações eletivas, para procedimentos cirúrgicos. Além disso, ainda são necessários mais estudos para determinar a necessidade de balonete, ou se esse protótipo poderia ser desenvolvido em formato cônico, podendo eliminar o Por se tratar de um estudo utilizando um software computacional, algumas variáveis, como temperatura corporal e pressão ajustada, podem gerar variações nos valores medidos de fluxo de entrada e saída, quando comparados a um estudo de bancada.A fluidodinâmica utilizando um software sugere que a fusão de 22cm iniciais de um tubo endotraqueal com 10,0mm de diâmetro e os 12cm terminais de um tubo endotraqueal com 6,0mm de diâmetro pode correlacionar-se com tubo endotraqueal convencional de 7,5mm em relação ao fluxo médio e resistência calculada de vias aéreas.A eliminação dos tamanhos de tubo endotraqueal convencional de diâmetros 6,0mm, 6,5mm, 7,0mm e 7,5mm pode gerar redução nos custos de serviços médicos. Como essa análise não foi parte integrante dos objetivos deste trabalho, estudos futuros são necessários para determinar essa estimativa.Ter um tubo endotraqueal de tamanho único pode ser benéfico, já que poderia facilitar a intubação orotraqueal com a utilização de um tubo endotraqueal fundido de 10,0/6,0mm, sem causar aumento da resistência calculada de vias aéreas. No entanto, este é apenas um estudo de simulação computacional.


PET imaging with both probes was conducted in patient-derived xenograft (PDX), subcutaneous and orthotopic, PDAC mouse models, and in a cancer cell line (CAPAN-2)-derived xenograft (CDX) model. Both radiolabelled tracers were successful in identifying, by means of PET imaging techniques, tumour tissues expressing MT1-MMP although they did so at different uptake levels. The 89Zr-DFO-LEM2/15 probe showed greater specific activity compared to the 68Ga-labelled peptide. The mean value of tumour uptake for the 89Zr-DFO-LEM2/15 probe  was 25–30 times higher than that of the 68Ga-DOTA-AF7p ones. Tumour/blood ratios (1.13 ± 0.51 and 1.44 ± 0.43 at 5 and 7 days of 89Zr-DFO-LEM2/15 after injection) were higher than those estimated for 68Ga-DOTA-AF7p probes (of approximately tumour/blood ratio = 0.5 at 90 min after injection). Our findings strongly point out that (i) the in vivo detection of MT1-MMP by PET imaging is a promising strategy for PDAC diagnosis and (ii) labelled LEM2/15 antibody is a better candidate than MT1-AF7p for PDAC detection.Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the deadliest cancers for which optimal diagnostic tools are still greatly needed. Identification of PDAC-specific molecular markers would be extremely useful to improve disease diagnosis and follow-up. MT1-MMP has long been involved in pancreatic cancer, especially in tumour invasion and metastasis. In this study, we aim to ascertain the suitability of MT1-MMP as a biomarker for positron emission tomography (PET) imaging. Two probes were assessed and compared for this purpose, an MT1-MMP-specific binding peptide (MT1-AF7p) and a specific antibody (LEM2/15), labelled, respectively, with  In addition, the desmoplastic reaction associated with PDAC may cause the malignancy to show up as a hypometabolic lesion under this imaging technique iodophenyl) maleimide ([123/125I]IPM) for SPECT/CT imaging in HT1080-tumour-bearing mice (HT1080 is a human fibrosarcoma cell line known to highly express MT1-MMP); the radiolabelled probe was therefore similar to DOTA-AF7p but with IPM instead of DOTA. Kondo et al. [99mTc]-(HYNIC-AF7p)(tricine)(TPPTS) for in vivo SPECT imaging of breast cancer. They found a highly tumour uptake of the probe (2.18 ± 0.11%ID/g at 2 h post-injection) in MDA-MB-231-tumour-bearing mice. These differences in the uptake of the AF7p tracer can be due to many parameters playing a role in targeting such as the diverse density of MT1-MMP in the tissues, the affinity of peptide after labelling, the rate of tracer delivery, the vascular permeability of tumours, and the interstitial pressure among others [The tumour uptake of ectively . This isg others .68Ga-DOTA-AF7p-1 and 68Ga-DOTA-AF7p-2  at 90 min after injection . As we did not find any statistically significant differences between the two radiolabelled peptides tested in CAPAN-2-bearing tumour mice, we decided to carry out our PET/CT studies after injecting 1.9 ± 1.8 MBq (n=17) of 68Ga-DOTA-AF7p-1. in vivo imaging, we also assessed the overexpression of MT1-MMP in patient-derived tumours by means of immunohistochemistry making use of the LEM2/15 antibody. Immunohistochemistry analyses revealed that MT1-MMP localized to the membranes of pancreatic ductal cells while no expression was detected in the stromal cells (Patient-derived xenografts (PDXs) are typically generated by the subcutaneous implantation of fresh, surgically derived human tumour material into immunodeficient mice. PDXs retain stably molecular, genetic, and histopathological features of their originating tumours and therefore are currently the only model system able to incorporate directly the vast interpatient and intratumour heterogeneity inherent to human cancer . With thal cells .in vivo volumetric measurements of subcutaneous tumours in mice [3 to 208 ± 137 mm3 along the whole duration of the PET imaging study  and it remained at this level throughout the time course: 0.227 ± 0.142 (n=8), 0.260 ± 0.047 (n=6), and 0.185 ± 0.082 (n=10) %ID/g tumour at 5, 8, and 14 days, respectively (68Ga-DOTA-AF7p-1 was of 0.221 ± 0.096%ID/g tumour (n=32). This is in good agreement with the results reported by Kondo et al. [68Ga-DOTA-AF7p-1 after injection in PDX  was higher than that measured in CAPAN-2-bearing tumour mice , demonstrating that for the PET visualization of the radiolabelled probe, the PDX model is better one than the CAPAN-2-derived model.Each mouse carried two patient-derived tumours implanted bilaterally subcutaneously. We used CT to evaluate volume growth; Jensen et al. had demonstrated that microCT was a more accurate than other methods  for the  in mice . All micng study . Althougour size . At the ectively . The meao et al.  in theirn=16 versus 4.03 ± 1.73, n=17 resp.).It should be noted that in addition to the tumour, kidneys and bladder also showed signals, indicating that the tracer is mainly excreted through the renal route . The mea68Ga-DOTA-AF7p uptake in pancreatic tumours would be required if we were to carry out further studies. A possible strategy could be to increase the affinity of the radiolabelled probe for MT1-MMP inserting a spacer linker that imposes a separation between the bifunctional chelating agent (DOTA) and the peptide, as it has already been suggested by several other authors [We are aware of, at least, one limitation in our research, that is the relatively low tumour accumulation level of both radiolabelled peptides, which was also occurred in the study of Kondo et al. . A setup authors , 31. Our89Zr and employed in the in vivo preclinical imaging of glioblastoma MT1-MMP+ tumours [89Zr-DFO-LEM 2/15 small-animal PET was conducted on mice harbouring xenografted tumours at opposite flanks with CAPAN-2 cells (n=4) after injection of 0.55 ± 0.01 MBq (n=4). n=7) at 1, 3, 5, and 7 days after injection, respectively, in CAPAN-2 tumours  was 25–30 times higher than that for the 68Ga-DOTA-AF7p ones. Sections of tumours immunostained for MT1-MMP confirmed the expression of this protein in the tumour cells but not in the stromal cells , readily labelled with  tumours . 89Zr-DF tumours . The meaal cells  as we haal cells .89Zr-DFO-LEM 2/15 after injection, resp.) (68Ga-DOTA-AF7p probes (approximately tumour/blood ratio = 0.5 at 90 min after injection). These results indicate that the visualization and quantification of the tumour uptake by noninvasive PET imaging is better with 89Zr-DFO-LEM 2/15 than it is with 68Ga-DOTA-AF7p. The difference in signal between tumour and normal tissue in the context of nuclear medicine imaging depends upon the tumour-localizing agent. Tumour-to-background ratios can be anything from 1 : 1 to >10 : 1 when using radiolabelled antibodies [Tumour/blood ratios in CAPAN-2 tumours increased lightly with time  . These vtibodies . Thus, t89Zr-DFO-LEM 2/15 at 1, 3, 5, and 7 days after injection was 8.84 ± 2.56, 9.83 ± 2.59, 9.46 ± 1.30, and 7.08 ± 3.30%ID/g, respectively. Lastly, bone uptake was low and did not increase over time, in support of the notion of the in vivo stability of the radioimmunoconjugate ; the 89Zr-DFO-LEM 2/15 uptake was 2.75 ± 0.44, 2.78 ± 0.61, 2.77 ± 0.58, and 2.46 ± 0.19 at 1, 3, 5, and 7 days after injection, respectively  as described above, the radioimmunoconjugate was prepared and injected  to the subject mice once the presence of tumours was confirmed by ultrasonography, and serial PET imaging was performed at 1, 2, 4, and 7 days. 89Zr-DFO-LEM2/15 was also injected in mice free of tumours (n=2) used as a control group. The PET images presented in 89Zr-labelled mAb LEM2/15. Tumour uptake increased over time, from 5.7 ± 1.4%ID/g at 1 d to 7.7 ± 2.5%ID/g at 7 d in PDX mice, whereas the accumulation in pancreas of control mice increased from 2.3 ± 0.3%ID/g at 1 d to 4.1 ± 0.1%ID/g at 7 d  and a MT1-MMP-specific binding peptide (MT1-AF7p) with


Perioperative fluid overload is an important modifiable risk factor for adverse outcomes after colorectal surgery. This study aimed to define critical thresholds for perioperative fluid management and postoperative weight gain for patients undergoing elective laparoscopic colorectal surgery.This was an analysis of consecutive elective laparoscopic colorectal resections at Lausanne University Hospital from May 2011 to May 2017. Main outcomes were overall, major (Clavien–Dindo grade IIIb or above) and respiratory complications, and postoperative ileus. Thresholds regarding perioperative fluid management and postoperative weight gain were identified through receiver operating characteristic (ROC) analysis and clinical judgement. Independent risk factors for all four outcomes were assessed by multinominal logistic regression.Overall and major complications occurred in 210 (36·2 per cent) and 46 (7·9 per cent) of 580 patients respectively. Twenty‐three patients (4·0 per cent) had respiratory complications and 98 (16·9 per cent) had postoperative ileus. Median length of hospital stay was 5 (i.q.r. 3–9) days. Based on respiratory complications, thresholds for perioperative intravenous fluid administration (postoperative day (POD) 0) were set pragmatically at 3000 ml for colonic  0·63)) and 4000 ml for rectal (AUROC 0·79) procedures. Postoperative weight gain of 2·5 kg at POD 2 was predictive of respiratory complications. Multivariable analysis retained perioperative intravenous fluid administration over the above thresholds as an independent risk factor for overall (odds ratio (OR) 2·25, 95 per cent c.i. 1·23 to 4·11), major  and respiratory  complications. Weight gain above 2·5 kg at POD 2 was identified as a risk factor for respiratory complications  and ileus .Perioperative intravenous fluid and weight thresholds were associated with postoperative adverse outcomes. These thresholds need independent validation. Stringent perioperative fluid management is a key component of enhanced recovery after surgery (ERAS) programmes, challenging traditional care schemes in many waysThe present study aimed to identify critical thresholds regarding perioperative intravenous fluid management and postoperative weight gain after elective laparoscopic colonic resections to facilitate guidance in daily clinical practice.Consecutive patients undergoing elective laparoscopic colorectal resections over a 6‐year period (May 2011 to May 2017) at Lausanne University Hospital were included. Patients were treated within a standard ERAS pathway over the study intervalThis study was considered an institutional quality improvement project; data extraction was approved by the Institutional Review Board . The study was conducted in line with the declaration of Helsinki and STROBE criteriaThree parameters were assessed: total intravenous administration on the day of surgery (postoperative day (POD) 0), comprising liquids administered during surgery  and those administered after surgery until midnight of POD 0 (perioperative fluids), using data from chart review of anaesthesia protocols; the amount of intraoperative intravenous fluid administered (weight and duration‐adjusted volume: ml per kg per h), where balanced administration was definedEndpoints were overall complication rate (Clavien–Dindo grade I–V)Length of hospital stay and overall compliance with the ERAS protocol greater than 70 per cent in patients with complete data sets for all 22 preoperative, perioperative and postoperative ERAS itemsOptimal thresholds for each fluid‐related parameter  were assessed by receiver operating characteristic (ROC) curve analysis. ROC curves were calculated with the Statistical and Machine Learning Toolbox™ of MATLAB R2018a . Confidence intervals for the area under the curve (AUC) were calculated by bootstrapping on 1000 replicas. Optimal operating points were determined mathematically as those points that jointly optimized the sensitivity and specificity of each ROC curve. Finally, optimal clinical thresholds were defined considering the decisional criteria of high negative predictive potential, early diagnosis, specificity and practicability.2 test was used for comparison of categorical variables, whereas Student's t test was used to compare continuous variables. All statistical tests were two‐sided, and a level of 0·050 was used to indicate statistical significance.Descriptive statistics for categorical variables are reported as frequencies, and continuous variables as mean(s.d.) or median (i.q.r.) values. The χFluid thresholds, together with demographic and surgical risk factors that were significant in univariable analysis, were included into a multinominal logistic regression model to calculate adjusted odds ratios (ORs) for the four main outcomes .Data analysis was performed with SPSS® Advanced Statistics 22 .Table Table The final analysis included 580 patients Table . The conTable TableFig. Thresholds  for intraoperative fluid administration, perioperative fluids and postoperative weight gain on POD 1, 2 and 3 were calculated for the four outcomes of interest   and negative predictive (NPV) values are shown in Table Perioperative fluid administration and POD 2 weight gain above the defined thresholds were entered, together with demographic and surgical risk factors, into a multinominal logistic regression model Table . IntravePerioperative fluid administration greater than 3 litres for colonic and 4 litres for rectal procedures, and weight gain of more than 2·5 kg on POD 2 were associated with adverse outcomes after elective laparoscopic colorectal surgery. These thresholds may be used in future work to investigate optimal fluid administration.versus 5 per cent) was observed in the restrictive fluid group, although 50 per cent of patients were not treated within an enhanced recovery pathway and fluid restriction did not increase the risk of acute kidney injury in patients treated within an ERAS pathway. A recent large‐scale studyPerioperative fluid management is a key item of enhanced recovery protocols and part of the overall strategy designed to decrease the surgical stress responseIn the present study three parameters were explored to define critical thresholds. Weight and duration‐adjusted volume (ml per kg per h) was not a reliable predictor of postoperative complications in the present study and has not been retained in recent guidelinesROC curve analysis revealed the thresholds to be most significant for respiratory complications, which occurred in 4·0 per cent of the study population, similar to findings in other studies of laparoscopic surgeryFrom the clinicians' perspective, thresholds should be pragmatic, easily assessable and, ideally, highly predictive. Although a threshold for total perioperative fluid administration might help in intraoperative management and during early recovery (for instance the use of vasopressors to decrease further fluid administration), a weight gain threshold at POD 2 might serve as a useful point of reference on the surgical ward. In the authors' institution, care maps are used to facilitate and standardize care within the established enhanced recovery protocol. In patients who exceed the thresholds, subsequent preventive measures, such as fluid restriction, promotion of mobilization and diuretics, can be triggered.This analysis has limitations as a result of its design and because it reflects a single institution's experience. Independent validation of these results is needed. The definition of thresholds was done not only by statistical means  but also by clinical and pragmatic considerations, indicating some subjectivity. On that basis, the cut‐off values should be considered to provide quantitative guidance. Urine output, which might have impacted on postoperative weight gain, was not measured in the present study. The association between complications and fluid administration and weight gain cannot be seen as causal, as higher perioperative fluid administration might have reflected more difficult procedures or complications.Independent validation of the proposed thresholds and prospective evaluation of treatments for unintended fluid overload are now needed.The authors declare no conflict of interest.Table S1 Predictive values for perioperative fluid and weight gain at POD 2 thresholdsClick here for additional data file.