Predictive preclinical models play an important role in the assessment of new treatment strategies and as avatar models for personalised medicine; however, reliable and timely model generation is challenging. We investigated the feasibility of establishing patient-derived xenograft (PDX) models of high-risk neuroblastoma from a range of tumour-bearing patient materials and assessed approaches to improve engraftment efficiency.PDX model development was attempted in NSG mice by using tumour materials from 12 patients, including primary and metastatic solid tumour samples, bone marrow, pleural fluid and residual cells from cytogenetic analysis. Subcutaneous, intramuscular and orthotopic engraftment were directly compared for three patients.PDX models were established for 44% (4/9) of patients at diagnosis and 100% (5/5) at relapse. In one case, attempted engraftment from pleural fluid resulted in an EBV-associated atypical lymphoid proliferation. Xenogeneic graft versus host disease was observed with attempted engraftment from lymph node and bone marrow tumour samples but could be prevented by T-cell depletion. Orthotopic engraftment was more efficient than subcutaneous or intramuscular engraftment.High-risk neuroblastoma PDX models can be reliably established from diverse sample types. Orthotopic implantation allows more rapid model development, increasing the likelihood of developing an avatar model within a clinically useful timeframe. However, limited engraftment success rates, lengthy model establishment time and high maintenance costs all pose major challenges to the routine use of PDX models for personalised therapy.18 Optimising the methodology to improve engraftment efficiency is critical for meeting urgent clinical needs in the era of personalised medicine.Patient-derived xenograft (PDX) models are generated from the implantation of patient tumour sample directly into immunodeficient mice, and the subsequent passage of tumour material from animal to animal.21 and has been described in more recent accounts.28 Herein, we demonstrate the feasibility of developing high-risk neuroblastoma PDX models at a high success rate from multiple sources of tumour-bearing patient materials at both diagnosis and relapse and from primary and metastatic tumour sites, and for the first time, residual tumour cells from cytogenetic analysis. We also show that the engraftment time is influenced not only by the number of tumour cells inoculated but also by the site of inoculation, with orthotopic  engraftment allowing more rapid model generation than subcutaneous engraftment. Finally, we also describe some of the challenges in developing PDX models—the development of xenogeneic graft versus host disease in recipient mice and the proliferation of Epstein–Barr virus (EBV)-infected cells rather than tumour cells—and provide strategies for obviating these hurdles.Establishment of neuroblastoma PDX models from primary tumour samples was first reported over 25 years agoFresh tumour specimens were obtained from patients at the Sydney Children’s Hospital Network (SCHN) under approval by the SCHN Human Research Ethics Committee . The primary purpose for developing the PDX models was to establish and improve xenografting approaches, to develop models to enable future preclinical testing, including in the context of personalised medicine. The relapse samples were from patients enrolled in a pilot feasibility study (TARGET) for a subsequent personalised medicine clinical trial , however without intention to return data to the treating clinician. Informed parental or guardian consent was obtained for each patient. The initial diagnosis of neuroblastoma was confirmed for each patient by a consultant paediatric pathologist. Samples were collected and transported in L-15 medium  at room temperature within 2–8 h after surgery. Solid tumour sample dissociation was performed enzymatically using a human tumour dissociation kit and a GentleMACS dissociator  into a single-cell suspension. Smaller samples were engrafted as 2–5-mm fragments. Bone marrow samples were collected in EDTA tubes and mononuclear cells isolated by Ficoll separation. Depletion of T lymphocytes was performed with human CD3 microbeads and an autoMACS Pro (Miltenyi Biotec) prior to assessment by flow cytometry and the CD3-negative population used for inoculation. Cells from pleural fluid were isolated by centrifugation and washed with red blood cell lysis buffer prior to inoculation. Cells from cytogenetics culture were obtained after completion of diagnostic analyses, harvested in EDTA-trypsin and washed in PBS prior to inoculation.2 with 2–6 mice/cage, and under 12:12 light/dark cycles. Bedding, enviro dry and igloos were provided for environmental enrichment. Irradiated rat and mouse breeder cubes and water were provided ad libitum. All animal procedures were performed inside a biosafety cabinet under sterile conditions during the animal’s light time cycle. Dissociated tumour cells, bone marrow, pleural fluid and cytogenetic cells (0.1–10 million cells) were mixed in RPMI (Life Technologies) and growth factor-reduced Matrigel  at equal volume ratio and implanted subcutaneously using a 27-gauge needle  into the flank of female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice29 obtained from Australian Bioresources . For implantation of tumour fragments, mice were anaesthetised using a Stinger anaesthetic machine  with 2–2.5% isoflurane  and 800 mL/min oxygen. A 5-mm horizontal incision was made on the dorsum of the mice, 10 mm rostral to the base of the tail, to create a subcutaneous pocket. A 2–5-mm fragment of tumour dipped in growth factor-reduced Matrigel was inserted and the incision site was closed with a wound clip. Buprenorphine   was administered for analgesia. Tumour growth was measured by Vernier callipers at least twice a week. Mice were euthanised once tumour size reached 1000 mm3, calculated using the formula: Length × Width × Height/2, by CO2 overdose followed by cervical dislocation. Tumours were triaged for histological and molecular analyses, cryopreservation and expansion in secondary recipient mice. Failure to engraft was defined as no measurable tumour at maximum holding time (12 months) and was confirmed by necropsy.All animal studies were performed in accordance with the guidelines approved by the University of New South Wales Animal Care and Ethics Committee (ACEC 17/101B) and the requirements of the Australian Code for the Care and Use of Animals for Scientific Purposes. Animals were housed in a specific pathogen-free environment in Techniplast research animal cages with filter tops and floor area of 400 cm5, 1 × 105 and 1.35 × 105 cells per mouse for A6580, A6912 and A7560, respectively). Subcutaneous inoculation, as well as anaesthesia and analgesia for intramuscular and orthotopic engraftment were performed as described above. For intramuscular engraftment, a 10-mm midline dorsal skin incision was made, 15 mm rostral to the base of the tail.30 Cells suspended in a mixture of 10 μL of RPMI and 10 μL of Matrigel were injected into the paraspinal muscle and the incision closed with 9-mm wound clips . Orthotopic  engraftment was performed as previously described.31 Briefly, a 15-mm skin incision was made adjacent to the caudal border of the spleen and the superior tip of the left kidney, followed by a 10-mm incision into the abdominal wall to expose the left adrenal gland. Cells suspended in a mixture of 10 μL of RPMI and 10 μL of Matrigel were injected into the fat pad surrounding the left adrenal gland. The abdominal wall was closed with a polyglactin-coated Vilet absorbable suture  and the skin incision was closed with wound clips. Mice were monitored daily for 7 days post surgery, and wound clips removed on day 7 post surgery. Intramuscular engraftment was assessed as described above. Orthotopic tumours were assessed by weekly ultrasound imaging and with callipers upon killing to confirm imaging-based measurement. Mice were euthanised by CO2 overdose followed by cervical dislocation. Event-free survival (EFS) was determined as described previously.32For each of three patients, a single tumour sample was dissociated and inoculated simultaneously into randomised cohorts of NSG mice at subcutaneous, intramuscular or orthotopic sites. A fixed cell number per mouse was used for each inoculation based on sample availability (5 × 102) adjacent to the left adrenal gland and prewarmed Aquasonic Clear Ultrasound Transmission Gel  was applied to the area. Imaging was performed using VisualSonics Vevo 2100 . A linear-array transducer (MS-250) traversed the selected area, acquiring 2D images at ~100-μm intervals, which were reconstructed as a 3D image in a dynamic cube format and analysed using Vevo Lab 1.7.0 software . Tumour boundaries were manually outlined in parallel slices within the 3D image and the final tumour volume calculated as the sum of the outlined tumour boundary for each slice.Mice were depilated . Stains included haematoxylin and eosin (H&E), Mason trichrome, orcein elastic and Papanicolaou and Giemsa. Immunohistochemical (IHC) staining included NB84 , synaptophysin , CD56 , PHOX2B , CD45 , CD20  and CD3 . For EBV-encoded RNA in situ hybridisation (EBER-ISH), slides were stained using BOND EBER probe (Leica). Images were captured using an Olympus BX53 light microscope and CD73 camera with CellSens software.TMgDNA Tissue Miniprep System  or Allprep DNA/RNA/Protein Mini Kit . For copy-number analysis using single-nucleotide polymorphism (SNP) arrays, DNA samples were processed using the Illumina InfiniumOmni2.5–8 according to the manufacturer’s guidelines to interrogate ~2.5 M SNP loci . Standard clustering and genotyping were performed using GenomeStudio v2.0.3  using default settings to collect SNP calls. Regions of copy-number variations (CNV) were called using cnvPartition2.0 with default parameters. B-allele frequency (BAF), log R ratio (LRR) and CNV plots were generated using package karyoploteR within the R statistical software package. CNV was determined as a function of BAF and LRR as previously described.33 Patient CNV data were also validated against the clinical data obtained through routine diagnostic DNA microarrays (Affymetrix Cytoscan 750K). Short-tandem repeat (STR) profiling results were analysed using PowerPlexR 18D System at the Kinghorn Centre for Clinical Genomics .DNA was extracted from patient and PDX tumours using the ReliaPrepInfiltration of human lymphocytes into mouse liver and spleen was determined by staining with APC mouse anti-human CD45 and FITC rat anti-mouse CD45. T-lymphocyte-depleted samples were analysed using PE mouse anti-human CD3 and APC mouse anti-human CD45. B lymphocytes in PDX tumours were stained with PE mouse anti-human CD19. For each sample, red blood cells were lysed by BD FACS lysing solution. PE mouse anti-human GD2 staining was used to determine the human neuroblastoma cell content in PDX tumours. All reagents were from BD Biosciences . Data were acquired with the LSRFortessa (BD Biosciences) and analysed with FlowJo software.Dissociated patient and PDX-tumour cells were seeded on 384-well plates at 2000 cells/well in 25 μL of Iscove’s Modified Dulbecco’s Media, 20% foetal calf serum and 1× insulin–transferrin–selenium (Life Technologies). Human neuroblastoma cell content in the PDX tumour was determined by flow cytometry. After 72 h, cells were treated with a library of 165 anticancer drugs. After a further 72 h, Cell-titer glo 2D (Promega) was added to measure ATP as a surrogate for cell viability. Cell viability as a function of drug concentration was plotted with a three-parameter sigmoidal dose–response curve to determine area under the curve (AUC) values. Correlation of drug sensitivity  in patient tumour cells compared with that for PDX-tumour cells was assessed using Pearson’s correlation analysis.3 tumour was 107 days (range 33–210 days). For the remaining samples, there was no evidence of engraftment after 12 months. The established models represented a wide patient age range at the time of diagnosis (0.3–7.5 years) and were derived from both MYCN-amplified4 and non-amplified6 samples. Engraftment at diagnosis was associated with poor outcome  and patients at the point of relapse from either Sydney Children’s Hospital Randwick or Children’s Hospital Westmead (five patients) Table . For twoEach of the established PDX models was authenticated as being derived from the donor tumour specimen by STR profiling. In each case, the match was 100% across the 18 amplified loci. Morphologically, all ten established subcutaneous xenografts recapitulated the features of the patient tumours from which they were derived (Supplementary Table r = 0.84–0.99) than for diagnosis models (r = 0.035–0.82). The PDX models generally conserved the established prognostic molecular markers and recurrent aberrations observed in the original tumour, including chromosome 1p loss and 17q gain, MYCN and ALK amplification and loss of PHOX2B, TP53, NF1 and ATRX , despite confirmation of sample identity by STR profiling (100% match) and histologic features that were consistent with high-risk neuroblastoma. Interrogation of a previous SNP array (Affymetrix Cytoscan 750K array) conducted on this patient sample for clinical care revealed that MYCN amplification and the major chromosomal losses  and gains , were all present in a tumour population that comprised 50% of cells  with even fewer differences in the PDX from cytogenetics culture (r = 0.95)  and the second from cytogenetics culture of the fresh tumour material (CCI-NB06-RMC). Compared with the original patient tumour, minor differences of chromosome 1p loss and 18q gain were observed in the PDX derived from fresh tumour material (95) Fig. . The higDespite the high success rate for subcutaneous engraftment of relapse samples, the success rate for diagnosis samples was below 50%, and the relatively long engraftment time poses a barrier to the use of these models in personalised oncology. To determine whether engraftment at alternative anatomical sites could increase engraftment efficiency, tumour cells from three neuroblastoma patients  were simultaneously inoculated into three cohorts of mice, subcutaneously, intramuscularly or orthotopically , and monitored for engraftment and tumour growth. In each case, the tumour sample was already known to engraft (Table 3 tumour) after 230 days. Intramuscular engraftment was faster than subcutaneous engraftment in one model (A6580), but slower in another (A6912). To exclude the possibility of bias based on different tumour volume measurement approaches, tumour volumes for all orthotopic xenografts were also obtained at endpoint by using callipers and compared with ultrasound analysis. The two measurement approaches yielded equivalent tumour volumes  led to alopecia and reddening of skin around the ears and eyes in the recipient mice, consistent with xenogeneic graft versus host disease (GvHD). Postmortem histopathological analysis of the liver from mice inoculated with each sample was also consistent with GvHD Fig. . Mild efFlow cytometric analysis of mononuclear cells isolated from liver and spleen of the mice indicated 55% and 36% CD45-positive human leucocytes in the A7167 mouse liver and spleen, respectively, and 95% CD45-positive human leucocytes in the A5723 mouse liver Fig. . To precr = 0.71).An attempt to generate a xenograft model from a pleural fluid specimen (patient A7012) containing both metastatic neuroblastoma cells and inflammatory cells, including small lymphocytes and neutrophils Fig. , producer = 0.9018, P < 0.0001, Fig. To further assess the biological similarly of primary patient cells and those expanded via mouse engraftment, a high-throughput screen of 165 anticancer drugs was performed on dissociated cells from both the A6580 patient sample and the first passage of the cognate PDX (CCI-NB06-RMT) under identical conditions Fig. . Prior t27 These models are predictive of clinical outcomes and therefore are an excellent platform for preclinical drug testing and as avatars for personalised medicine.11 This study extends previous neuroblastoma PDX model establishment reports28 by demonstrating that PDX models can also be developed from diagnostic cytogenetic culture without additional loss of fidelity, that more rapid engraftment can be achieved with orthoptic implantation and that additional sample processing can circumvent loss of models by xenogeneic GvHD.PDX models are known to preserve many of the key biological properties of the tumours from which they were derived and remain stable across passages.26 in which neuroblastoma samples were engrafted para-adrenally with a success rate of 24% at diagnosis (9 of 39) and 33% at relapse (1 of 3). Several factors might contribute to the variation of engraftment rate between these studies, including the source of patient material, time to engraftment, number of cells inoculated and engraftment technique. In an earlier study,21 the subcutaneous engraftment rate of PDX from neuroblastoma patients at the time of diagnosis was 34% (20 of 58) for all patients attempted, and 45% (15 of 33) from INSS stage IV disease, which is similar to the current study. We found that histologic and molecular features are generally preserved in high-risk neuroblastoma PDX models commensurate with previous reports.28 The high success rate for samples from cytogenetics culture by comparison with samples from other patient sources that failed to engraft, strongly suggests that these materials should be accessed for model development where possible. While one PDX model (CCI-NB04-DML from A7167) developed from a lymph node metastasis via cytogenetics cells showed very poor correlation with the originating tumour material based on the CNV profile, further analysis of a clinical SNP array for these samples suggests clonal selection in the PDX, and good concordance with the expanded clone in the original patient material. More generally, evolution and copy-number alteration that occurs during development of PDX models might provide explanation for other differences.35Our subcutaneous engraftment success rates of 44% (4 of 9) for diagnosis patients and 100% (5 of 5) for relapse patients compare favourably with a recent reportscid mutant strains and do not develop spontaneous thymoma,36 which are important considerations when engraftment time is long, as is frequently the case for neuroblastoma models. While the absence of functional NK cells renders NSG mice highly susceptible to xenogeneic GvHD development,37 we show that this can be avoided by T-lymphocyte depletion of the patient sample.With further optimisation, other potential samples, such as pleural fluid and lymph node metastasis biopsy, can be used as valuable resources for PDX development, increasing engraftment success rate per patient, an essential advance for implementing personalised treatment in this childhood malignancy. Avoiding the loss of engrafted samples due to mouse pathologies is also critical for reliable model development and judicious choice of recipient mice and sample-processing approaches is essential. Highly immune-deficient NSG mice are advantageous for tumour engraftment, have significantly longer life spans than other 43 and the incidence rate in NSG mice has been reported to be as high as 32%.41 In healthy individuals, EBV infection is benign with reactivation and subsequent proliferation of EBV-infected lymphocytes suppressed by immune detection and response,44 whereas in an immunodeficient patient, EBV can transform B cells into the growth phase and trigger the lymphoproliferative disease.45 Similarly, the lack of immunosurveillance in recipient mice allows the proliferation of the EBV-infected lymphocytes contained in the patient.42 PCR-based detection of the EBV BamHI W region, a major internal repeat in EBV genome, in the patient specimen to be engrafted, has been reported to correlate with EBV-related lymphoid proliferation in the derivative PDX and may allow identification of samples requiring lymphocyte depletion.44 Alternatively, atypical lymphoid proliferations might be obviated by administration of rituximab, an anti-CD20 antibody, as the rate of lymphoma incidence in ovarian cancer PDX models has been reported to decline from 11.1 to 1.88% after the addition of rituximab.46 Aside from prevention, a robust approach to identify atypical lymphoid proliferation is essential to maintaining xenograft validity and mandatory prior to additional clinical assays, such as drug screening, being performed on the xenograft material.Development of EBV-associated atypical B-lymphoma proliferation or EBV-related B-cell lymphoma has been previously described after attempted xenografting of other human tumour types in immune-deficient mice30 or a greater capacity for angiogenesis.31 While enabling a shorter engraftment time, orthotopic inoculation is more technically challenging than subcutaneous inoculation and animal monitoring typically requires in vivo imaging,48 which can be particularly labour intensive for the frequent monitoring required in preclinical testing.48 Our observations that orthotopic, intramuscular and subcutaneous xenografts are equally representative of the donor tumour suggest that engraftment approaches can be selected based on utility.A major challenge for the use of high-risk neuroblastoma PDX models in personalised medicine is the development, validation and application of models in a timeframe that can still inform individual patient treatment. Approaches that shorten engraftment time or increase engraftment success rates without compromising model fidelity are essential to make this approach broadly applicable. Our observation that for each of three patient samples, orthotopic adrenal engraftment was substantially faster than either subcutaneous or intramuscular engraftment suggests one approach to reducing engraftment time. The reasons for more rapid adrenal engraftment are not clear; however, the adrenal gland is the most common site of origin for primary neuroblastoma. Alternatively, previous studies have suggested that non-subcutaneous sites may provide greater protection of the engrafted cells against hypothermic insult,49 This study demonstrated that PDX-derived cells were dependable alternatives for screening high-risk neuroblastoma, with ex vivo drug sensitivity of primary patient cells and PDX-expanded material being highly correlated.In addition to in vivo preclinical testing, limited patient material expanded as a PDX model can be used as a primary short-term culture for high-throughput ex vivo testing of drug sensitivity, including for combination chemotherapy.In summary, we identify dependable engraftment approaches that increase the likelihood of developing informative preclinical models for individual high-risk neuroblastoma patients within a clinically relevant timeframe and reliably expand limited patient material for ex vivo and in vivo drug testing.Supplementary materials


Solitary fibrous tumour of the pleura (SFTP) arising in a cavity is extremely rare. A 66‐year‐old Japanese male presented with an abnormal shadow on his chest X‐ray. Chest computed tomography showed a cavity of approximately 18 mm in diameter between the upper and lower lobes that contained a solid nodule within. Under the thoracoscope, the peduncle cystic tumour was removed with sufficient surgical margin. Macroscopically, a tumour of about 15 mm in diameter arose in the cystic cavity. Immunohistochemical stains were positive for CD34, bcl‐2, and signal transduction and activator of transcription 6 (STAT6) but negative for smooth muscle actin (SMA), desmin, and epithelial membrane antigen (EMA), and a diagnosis of SFTP was made. The patient remains well without recurrence or any complications at two and a half years after the operation. SFTP should be considered when a tumour arises in a cavity existing in an interlobar space. It is important to determine whether the tumour is pedunculated or sessile during surgery and to perform the appropriate surgical procedure. Solitary fibrous tumour of the pleura (SFTP) is a rare neoplasm. Moreover, cavity formation associated with SFTP is extremely rare. To our knowledge, only one such case has been previously reported. Solitary fibrous tumour of the pleura (SFTP) is a rare neoplasm that accounts for less than 5% of primary pleural tumours and has an annual incidence of 2.8 cases per 100,000 individuals [A 66‐year‐old non‐smoking Japanese male presented with an abnormality found on a chest X‐ray performed during an annual check‐up. The patient had no past medical history and an unremarkable family history. His chest X‐ray showed a well‐defined 15 mm × 11 mm nodule in the left lower lung field Fig. . Chest cUnder the thoracoscope, the pedunculated cystic tumour arose from the S4 area and was removed with sufficient surgical margins. Macroscopically, the tumour arose in the cystic cavity and was approximately 15 mm in diameter Fig. . MicroscBaek et al. reported a case similar to the one reported here in a 58‐year‐old non‐smoking woman with an air‐containing SFTP located in the right major fissure . They sp2 is the most reliable indicator of aggressive behaviour [Although most of the SFTP are benign, up to 10% are malignant [Mahesh et al. reported a case of recurrence in a benign SFTP more than 10 years after resection . Moreovede Perrot et al. proposed a staging system and algorithm for management and follow‐up of SFTP in 2002 . The staTapias et al. proposed a scoring system to predict recurrence after SFTP resection in 2013 . They coSFTP, although rare, should be considered in cases of a tumour arising in a cavity existing in an interlobar space, especially with a gradually thickening cyst wall. It is important to determine whether the tumour is pedunculated or sessile during surgery and to perform the appropriate surgical procedure.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


This case has interesting images of huge hydro pneumoperitoneum. It is a rare view in the medical practice and a good point to learn one of the complications of perforated duodenal ulcer and failure of omental patch operation. We reported a case of acute abdominal pain with hydro pneumoperitoneum that appeared as an air fluid line in X-ray. The patient had an omental patch surgery. Two days after the operation he got severe abdominal pain. The X-ray images showed significant huge hydro pneumoperitoneum. He underwent urgent surgical intervention for exploration that detected a large amount of gases, a biliary free fluid and a leak from duodenal ulcer. Omental buttressing was then performed. A hydro pneumoperitoneum refers to free air and fluid in the peritoneal cavity . WhereasA 47-year-old man presented to the emergency department with acute abdominal pain and vomiting. On examination, vital signs were stable, chest was clear and the abdomen was rigid and there was tenderness. Focused bed side ultrasound showed large amount free fluid. Chest X-ray showed an average amount of air under diaphragm with air fluid line . The patPneumoperitoneum is one of complications of perforated duodenal ulcer . The airet al. documented a 12% rate of ulcer recurrence after omental patching and a 23% incidence of recurrent symptoms within 44 months  were treated by omental patching alone, Smith We have reported the case of a significant huge hydro pneumoperitoneum which is an unusual presentation in perforated duodenal ulcer. Omental patch repair does not correct the underlying process that causes perforation, and ulcers may recur. Emergency physicians and surgeons should be aware of unusual presentation of duodenal ulcer and other diseases. Omental patch operation could induce huge hydro pneumoperitoneum if it fails and the patient had a presentation of hydro pneumoperitoneum.The authors declare no competing interests.


The aim of this study is to compare two different strategies of perioperative hemodynamic optimization: one based on optimization of preload using dynamic parameters of fluid-responsiveness and the other one based on estimated oxygen extraction rate  seems to be not adequate for every patient, a personalized therapy is likely more appropriate. Following this concept, dynamic parameters of fluid-responsiveness allow to titrate fluid administration aiming CO increase but avoiding fluid overload. This approach has the advantage of personalized fluid therapy, but it does not consider if CO is adequate or not. A protocol based on O2ER considers this second important aspect. Although positive effects of perioperative GDT have been clearly demonstrated, currently studies comparing different strategies of hemodynamic optimization are lacking.As a predefined target of cardiac output (CO) or oxygen delivery (DONCT04053595. Registered on 12/08/2019.ClinicalTrials.gov,  The same authors showed that the incidence of organ failure and mortality were reduced when oxygen debt was rapidly compensated using a protocol of hemodynamic optimization aiming to reach the same hemodynamic targets recorded in survived patients ) .p value of F-test < 0.05 will be considered for variable inclusion. The amount of multicollinearity in the model will be estimated by the variance inflation factor (VIF). A VIF higher than 4 will be considered a sign of multicollinearity. Predicted R2 will be used to assess overfitting.Student t test or Mann–Whitney U test will be used to analyzed secondary outcomes as appropriate. Mortality between groups will be analyzed using the Kaplan-Meier method. The Log Rank test will be used to evaluate the statistical significance. Cox regression model using forward selection method for entering explanatory variables will be used to perform multivariate analysis. The age, type of surgery, ASA-PS classification and comorbidities will be considered as variables. A p value is calculated for testing the hypothesis HR = 1 versus the alternative HR > 1 . If the p value is < 0.0394 at a monitoring time, then the trial would stop with the conclusion that non-inferiority cannot be claimed [An interim analysis will be performed by independent statistician when 50% of information will be available (100 patients recruited). To monitor for harm or futility, the one-sided  claimed , 19. Sto claimed .p value < 0.05 is considered statistically significant.For each statistical test, a 2 seems to be not adequate for every patient, a personalized therapy is likely more appropriate. Following this concept, dynamic parameters of fluid-responsiveness (PPV and SVV) allow to titrate fluid administration aiming an increase of CO as a consequence of preload optimization but avoiding fluid overload. This approach has the advantage of personalized fluid therapy, but it does not consider if CO is adequate or not because preload optimization occurs independently from parameters of tissue perfusion and oxygen consumption. A protocol based on O2ER considers this second important aspect. An O2ER higher than a predefined cutoff is reflecting an inadequate DO2 to tissue and consequently an inadequate CO. This condition, but not fluid-responsiveness per se, is the trigger to start hemodynamic optimization. Moreover, in this contest we believe that CVP may be useful to limit fluid administration independently from PPV or SVV value. In fact, CVP depends on several conditions including preload status but also on cardiac function and ventricular compliance. Thus, this trial aims to demonstrate that this latest strategy of perioperative hemodynamic optimization is not inferior to the former in term of postoperative complications, but it may be responsible for a lower fluid administration. We decided to conduct a non-inferiority study because PGDT is routinely applied in this kind of patients due to the demonstrated benefits on outcome. Thus, small difference in reduction of postoperative complication rate between two different PDGT protocols seems reasonable.The role of perioperative hemodynamic optimization has been clearly shown by literature and the current spreading of mini-invasive monitoring systems able to estimate CO permits an easier application of these protocols than occurring in the past when only invasive techniques (e.g. pulmonary artery catheter) were available. As a predefined target of CO or DOMethodologically, the main limitation of this trial is the inability to blindly administer the interventions. However, this point is not clinically resolvable in this research field. Moreover, if participation of several centers may increase enrollment rate and may potentially extend the applicability of results, on the other hands too few patients per center may increases heterogeneity.Protocol version 2.0 of 26.06.2019. At the date of manuscript submission, the patients’ recruitment process is ongoing. It is expected that the recruitment will be complete within December 2021. The results of this study have not already been published or submitted to any journal.Additional file 1. Participating centers list.


Fluid therapy is a rapidly evolving yet imprecise clinical practice based upon broad assumptions, species-to-species extrapolations, obsolete experimental evidence, and individual preferences. Although widely recognized as a mainstay therapy in human and veterinary medicine, fluid therapy is not always benign and can cause significant harm through fluid overload, which increases patient morbidity and mortality. As with other pharmaceutical substances, fluids exert physiological effects when introduced into the body and therefore should be considered as “drugs.” In human medicine, an innovative adaptation of pharmacokinetic analysis for intravenous fluids known as volume kinetics using serial hemoglobin dilution and urine output has been developed, refined, and investigated extensively for over two decades. Intravenous fluids can now be studied like pharmaceutical drugs, leading to improved understanding of their distribution, elimination, volume effect, efficacy, and half-life (duration of effect) under various physiologic conditions, making evidence-based approaches to fluid therapy possible. This review article introduces the basic concepts of volume kinetics, its current use in human and animal research, as well as its potential and limitations as a research tool for fluid therapy research in veterinary medicine. With limited evidence to support our current fluid administration practices in veterinary medicine, a greater understanding of volume kinetics and body water physiology in veterinary species would ideally provide some evidence-based support for safer and more effective intravenous fluid prescriptions in veterinary patients. Fluid therapy paradigms are constantly changing –3 due toThe recent discovery by the “Fluid Expansion as Supportive Therapy” (FEAST) study in African children whereby standard fluid boluses significantly increased 48 h mortality  took theFor decades, research in fluid therapy was constrained by the lack of effective methods to assess important effects and outcomes. Fluid dynamics and volume expansion effects have been investigated using conventional indicator dilution techniques , 18, bloFor the past 20 years, Robert G. Hahn and his research group have been studying and refining the concept of volume kinetics (VK), providing a research platform to understand how administration of various IV fluids affect body fluid compartments under different physiologic conditions. Intravenous fluids can now be studied like pharmaceutical drugs, leading to improved understanding of their time-volume effects or elimination half-life on plasma and interstitial fluid compartments , making The study of VK in companion animal species is unprecedented, and there is no scientific literature currently available. In response to the call for more scientific research to build an evidence-based foundation for veterinary fluid therapy, the purpose of this review is to introduce the basic concepts of VK, its current use in human and animal research, as well as its potential and limitations as a research tool for fluid therapy research in veterinary medicine.Pharmacokinetics and pharmacodynamics are complex disciplines that utilize advanced mathematical principles that practitioners sometimes find challenging to comprehend. However, these two branches of pharmacology are pivotal in understanding ways to enhance efficacy and decrease harm for any exogenous substance prescribed , thus crVd) is a proportionality constant between measured plasma concentration and the corresponding amount of substance within the body according to the state of substance disposition, i.e., at initial time 0 (Vd(c)), under steady-state conditions (Vd(ss)), pseudo-equilibrium conditions (Vd(area)), or at any given time (Vd(t)) ) . Volume  (Vd(t)) . Finally (Vd(t)) . The ter (Vd(t)) . Most su (Vd(t)) . Zero-or (Vd(t)) .The management of a substance by the body is an intricate process as the various components of “ADME” occur simultaneously, thereby constantly altering substance concentration in tissues and fluids . ApplicaCompartmental models are said to be deterministic because the model that best describes the PK of a substance is determined by the substance's concentrations in the blood . If a suThe merit of any model depends on how well it mimics the substance's concentration in the body. Typically, the simplest model that sufficiently predicts changes in substance concentration over time is selected . A conceThe conventional way to present PK output is to analyze each subject individually and report the mean parameter values for a group as the “typical parameter values” for that cohort. An alternative approach is to analyze all subjects in one single analysis while keeping track of the between-individual variability. The widespread adoption of such population modeling in human pharmacology, also known as non-linear mixed effects (NLME) modeling, provides a framework to characterize sources of variability in drug disposition and response using statistical models that account for between- and within- individual and experimental variation –58. IdenVolume kinetics (VK) is an adaptation of PK theory allowing insights into the effects of IV fluid on theoretical body fluid compartments –33, 55. When “ADME” concepts are put into the context of IV fluid administration, the absorption is instantaneous with a 100% bioavailability, metabolism is irrelevant, therefore leaving only distribution and elimination to be considered. The central concepts of both PK and VK analyses are therefore dependent upon the volume of distribution and clearance of an administered substance . As in PUnlike pharmaceuticals, water is the main component of both infusion fluids and plasma , 31, theVd) in conventional PK analysis is not easily applied to fluid therapy. Unlike conventional PK, where Vd is assumed constant as the volume of administered drug is often negligible, the volume of distribution of IV fluid (denoted by the symbol v) continually changes as a direct consequence of IV fluid administration. Therefore, the baseline output parameter, volume of expandable body fluid space (V), representing the volume of distribution of initial fluid infusion is estimated instead in VK  and the volume of expandable peripheral body fluid space (Vp). V can only be estimated from plasma dilution measurement and represents the functional fluid space actually expanded by IV fluid  and actual urine output measurement may also assist in final model selection  or bi-exponential (2-VOFS kinetic model) curve. The dilution-time profiles, sometimes augmented by urinary excretion , are entribution . Similarribution  and statribution , 62. Agrelection .TM    can be uVp), which became an issue with slow fluid elimination  are substituted by rate constants  and fractional plasma dilution (vc − Vc)  , 33 in tnfusions . The micstigated , 33.With the evolution of VK models over time, many parameters have been represented differently, and so it is important to review the literature with an eye to this evolution to not be confused by the changes in symbols and parameters. This is further complicated by the use of proprietary symbols and expressions between PK computer software and PK studies . This haV1 = Vc, V2 = Vt = Vp, v1 = vc, v2 = vt = vp, ki = R0, kb = Cl0 = k0, kr = Cl ≅ k10, and kt = Cld ≅ k12 and k21  would expand the volume of a central body fluid space (Vc) to a larger volume (vc) (vc − Vc) (Vp) which in turn expands to a greater volume (vp). These central and peripheral body fluid spaces attempt to retain their respective initial volumes to maintain homeostasis (Vc and Vp multiplied by the distribution clearance (Cld) in the clearance model  in the micro-constant model (k12) and peripheral to central intercompartmental rate constant (k21) akin to PK analysis  . As tranvc-VcVc) , 32 or avc − Vc) , 77, 78 eostasis , 32. Thece model , or the nt model , 77, 78.analysis , 77.Cl0 or kb) often pre-set between 0.3 and 0.5 mL/min in humans (Cl or k10) (vc − Vc) (Cl) or elimination rate constant (k10)  baseline water loss which is accounted for by zero-order elimination  . The ratvc-VcVc) , 32 or avc − Vc) , 77, 78,Vc, Cld (or k12 and k21), and Cl (or k10)  is then set to a fixed value determined by the total urinary excretion obtained. In this scenario, Cl is analogous to the renal clearance which is calculated by dividing the urine output by the integral of the plasma dilution-time curve, also known as the area under concentration-time curve (AUC), following the assumption that half of the Cl0 or k0 due to insensible fluid losses appears as urine (The unknown parameters in the 2-VOFS kinetic model that are estimated by non-linear least-squares regression are (or k10) . If urinuid loss , 60. Theas urine :The differential equations describing the micro-constant model for the 2-VOFS kinetic model are as follows :Cld is obtained as the product of Vc × k12 and Vp × k21 (Cl is the product of Vc × k10 (By comparing Equation (1) for the clearance model with Equation (3) for the micro-constant model, net Vp × k21 . SimilarVc × k10 .Cl : Cld (or k10 : k12 and k21) ratio. This reduces the time for full Vp expansion prior to elimination, therefore Vc and the partially expanded Vp fuse into a moderate size single body fluid space, allowing 1-VOFS kinetic model to sufficiently describe these dilution-time profiles  is thought to expand the volume of a single body fluid space (V) to a larger volume (v) (Cl0 or kb) and a dilution-dependent, primary renal excretion governed by a first-order elimination (Cl or k10) (V and Cl (or k10) (v − V) in the micro-constant model, is obtained from plasma dilution computed from serial Hb concentration. Similar to the 2-VOFS kinetic model, Cl0 and Cl can be estimated through VK modeling or calculated if urine output is available. The following differential equation describes the clearance model for the 1-VOFS kinetic model  . Elimina or k10) . The unk(or k10) . The fraic model , 79:In comparison, the differential equation that describes the micro-constant model for the 1-VOFS kinetic model is as below , 77, 78:The volumes of individual body fluid compartments are conventionally estimated using external indicator-dilution techniques. Some examples include but are not limited to, deuterium oxide  or radioAlthough exogenous indicator-dilution techniques are considered the conventional “gold standard” method for volume determination , 86, theIn 1987, an endogenous indicator-dilution technique using blood Hb concentration was introduced as an accurate method for estimation of BV changes following validation with the radio-iodinated serum albumin technique . EndogenV or Vc is directly proportional to plasma dilution ) is first obtained, then the total Hb mass corrected for sampling at any time (t), followed by the expanded BV corrected for sampling at any time (t)  :t) can be provided by Equation (11), and the BV expression can be easily transformed to PV using Equation (12):Changes in BV at any time ) and diluted Hb at any time (t) is written as The relationship between baseline Hb  . The lowThe degree of error introduced by the wide range of initial BV has been shown to be forgiving , howeverMCV(0)) and at any time (t) into Equation (13) above:When investigating IV fluids that alter plasma osmolality, for example hypertonic saline (HS) or glucose infusions, a correction for changes in mean corpuscular volume should also be considered. Mean corpuscular volume can be altered in humans following infusion of HS because of osmotically induced decreases in intracellular water, this has also been reported in dogs and cats . ChangesOsm(0)), which is ~295 mOsm/kg for humans (ft) can be obtained from Equation (16)  to the extracellular fluid space (~20% of BW) . Using sr humans , ~317 mOr humans , and ~30ion (16) , 68:ft should be entered as a linear function in the analysis process such that ft at each point in time is governed by the total amount of infused fluid   , 60, 68,% of BW)  and meas% of BW) . Howeverze of PV , 60, 68 o the PV .Vp in healthy adult humans was discovered to be 6–8 L (8–11% of BW)  , 68, 96,% of BW) . The Vp by fluid , 34, 96.by fluid . Some inby fluid , 98. Theto sheep . The pre than Vc , 32, 60 nowledge . So far,reported .Cld) or the intercompartmental rate constants  can be appointed the same value for bi-directional flow based on the assumption that fluid is equally prone to flow in either direction  that is estimated by non-linear least square regression should approximate the renal clearance (ClR), since the ultimate destiny of IV fluid is elimination via the kidneys  of infused fluid is by utilizing ClR which is obtained by dividing urine output with the AUC for the fractional plasma dilution or absolute volume expansion in the clearance or micro-constant model, respectively   was computed by VK modeling (The first n 80 kg) . Balancen 80 kg) . Urinarymodeling . This womodeling . Acetatemodeling .Over the years, VK analysis has provided some mechanistic explanations as to why and when patients are sensitive to large volumes of fluid . Slow flft).Volume kinetic analysis has also been used to describe the distribution and elimination of isotonic and hypertonic fluids within the body . When IVVc for HES in both series of experiments was 3.14 L (~5% of BW), close to the PV estimated by anthropometry, with a t1/2 of 2 h (t1/2 reported in the product monograph (Vc for acetated Ringer's solution averaged 4.88 L (~6.2% of BW) with a t1/2 of 88 min  in conscious sheep; however when anesthetized and mechanically ventilated, urinary excretion was markedly reduced  resulting in peripheral fluid accumulation  (kr) predicted by VK analysis (58.5 mL/min) was significantly different from the manual calculation using median urinary excretion (0.6 mL/min) when sheep were anesthetized and mechanically ventilated  . Howeverntilated . Estimatmulation . This lepresumed , 60, 109A subsequent experimental cross-over study by Connolly et al.  in 7 norVc was found to be proportional to the magnitude of plasma dilution regardless of infused volumes and rates, and elimination occurs via expansion of Vp when renal excretion fails to increase in proportion to the volume of infused fluid  to 327 ± 54 mL (7.5% of BW), and the rate of elimination  increased from 2.9 ± 0.5 to 5.9 ± 2.8 mL/min during early normotensive endotoxemia  was found to promote renal excretion of infused fluid at the expense of fluid distribution to the periphery thus limiting the volume expanding effects of the fluid infusion, while β1-adrenergic receptor agonist  had the opposite effect. The interaction between fluid administration and vasoactive drug  delivery was recently further explored in an experimental sepsis model in sheep  to the same extent as it did in healthy awake sheep  in a population of healthy awake sheep . Vasoactin sheep . Resultsin sheep  in that ke sheep  due to tke sheep . In addike sheep . The resTM IIR hematocrit monitor, In-Line Diagnostics, Kaysville, UT) that optically measured hematocrit along an extracorporeal circuit established between a central venous jugular catheter and a cephalic venous catheter to describe the real-time BV changes over 4 h, following rapid IV fluid administration  . Silversstration . This mostration . In thisstration . Althougstration . Similarstration , isotonistration .Following publication of this study by Silverstein and colleagues, IV fluid expansion and retention of differing resuscitative fluids have been widely disseminated in the veterinary literature and textbooks and generalized to the feline species. The use of a non-invasive continuous in-line hematocrit monitor in Silverstein's study was an innovative and unique method which obviated the need for repeated invasive blood sampling, the use of sophisticated mathematical modeling, or isotope administration to estimate BV. However, this method requires placement of a central venous catheter, heavy sedation or general anesthesia, and the use of an extracorporeal circuit which is not practical in the clinical setting, except for patients receiving renal replacement therapies.As mentioned previously, VK has not found widespread adoption in human medicine due to the complexity of these analyses requiring some mathematical and statistical background, combined with an understanding of biology, pharmacology, and physiology. Volume kinetic modeling and analysis involves specialized PK modeling software that utilizes distinctive PK nomenclature and programming languages which may not be intuitive to the general scientific audience. Data collection is simple and straightforward; however, data analysis and interpretation involve a steep learning curve for those who are unfamiliar with these concepts. Training courses and first-hand utilization of the PK modeling software would be able to accelerate this learning curve.The main restriction to extensive clinical use of VK is the requirement for repetitive invasive Hb sampling . SuccessMass balance technique using laboratory blood Hb changes has been widely used to estimate intravascular volume status , 138–140Recommendations from published VK studies include utilizing a safe yet adequate IV infusion volume, e.g., 20–25 mL/kg crystalloid fluid bolus over 30 min, to minimize “noisy” data and estimation errors associated with very small infusion volumes , 92. ManVolume kinetics is an innovative research method that is gaining recognition for its wealth of accumulated evidence in this new era where clinicians are searching for context-sensitive fluid therapy paradigms. Despite its limitations, the pharmacokinetics of IV fluid therapy is still an appealing concept that has the potential to serve as a new research tool in veterinary medicine to provide insights on the distribution and elimination of commonly prescribed IV fluids. This research method is feasible and deserves a thorough investigation in the companion animal species. The detailed time course of IV fluids within the body, volume expansion effect, efficacy, half-life (duration of effect), and body water physiology in different patient populations under various clinical conditions may allow for more evidence-based IV fluid therapy prescriptions for our veterinary patients. Until a less invasive method of serial Hb monitoring has been validated for veterinary patients, VK will likely remain as a fundamental research tool for modeling and simulation of IV fluid therapy.XY wrote the first draft of the manuscript. SB, AB, WM, and RH provided critical revision of the manuscript. All authors read and approved the final version of the manuscript to be published.The authors declare that the manuscript was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As percutaneous transthoracic needle biopsy (PTNB) of the lung is a well-established diagnostic method for the evaluating pulmonary lesions, evidence of safety based on representative data is limited. This study investigated the practice patterns of PTNB of the lung and assessed the incidence and risk factors of PTNB-related severe pneumothorax in Korea.We used a national-level health insurance database between January 1, 2007 and December 31, 2015. Patients who underwent PTNB of the lung were identified using procedure codes for organ biopsy, fluoroscopy, computed tomography, chest radiography, and lung-related diagnosis codes. The annual age-/sex-standardized rate of PTNB and the incidence of PTNB-related severe pneumothorax were calculated. We defined severe pneumothorax as the pneumothorax requiring intervention. The odds ratios of risk factors were assessed by a generalized estimating equation model with exchangeable working correlation matrix to address clustering effect within institution.A total of 66,754 patients were identified between 2007 and 2015. The annual age-/sex-standardized rate of PTNB per 100,000 population was 19.6 in 2007 and 22.4 in 2015, and it showed an increasing trend. The incidence of severe pneumothorax was 2.4% overall: 2.5% in men and 1.2% in women, and 2.6%, 2.7%, 2.1%, 2.1%, 1.9%, 2.4%, and 2.4% from 2009 to 2015. Older age (≥60), male sex, presence of chronic obstructive pulmonary disease, receiving treatment in an urban or rural area versus a metropolitan area, and receiving treatment at a general hospital were significantly associated with the risk of severe pneumothorax.Considering the increasing trend of PTNB, more attention needs to be paid to patients with risk factors for severe pneumothorax. Percutaneous transthoracic needle biopsy (PTNB) of the lung is a well-established diagnostic method for the pathologic evaluation of pulmonary lesions. It has been proven to be minimally invasive and effective, and is therefore recommended by the relevant guidelines in cases of peripheral lung cancer , 2. For Even though PTNB is considered to be relatively safe procedure, pneumothorax, pulmonary venous air embolism, hemoptysis, and death have been reported as complications related to PTNB , 11. PneThe published rates of pneumothorax as a complication of PTNB vary widely according to the study setting, and the majority of previous studies included fewer than 1,000 patients and were conducted using records from a single institution –15. As iTherefore, the purpose of this study was to evaluate the rate of PTNB, the incidence of severe pneumothorax as a complication of PTNB, and the risk factors for PTNB-related severe pneumothorax in the Korean population using a national-level health insurance database.The Korean National Health Insurance system was launched in 1977 and achieved universal coverage of the entire Korean population in 1989. Health insurance claims data, also known as Health Insurance and Review Assessment (HIRA) data, are publicly available for research after de-identification. The HIRA data are comprehensive, and contain general information , healthcare services , diagnoses, and outpatient prescriptions. Diagnoses were coded using the Korean Standard Classification of Diseases Version 6, which is based on International Statistical Classification of Diseases 10th revision (ICD-10) codes. We obtained claims data for patients who underwent deep-organ needle biopsy from January 1, 2007 to December 31, 2015.As the HIRA procedure codes do not contain an exclusive code for PTNB, but only a code for deep-organ needle biopsy, which includes PTNB, we defined PTNB using the HIRA procedure codes for the imaging methods that accompanied PTNB, the type of medical institution, specialty, and diagnosis codes. The patients were defined as having undergone PTNB when the following eligibility criteria were met on the same date: 1) deep-organ needle biopsy; 2) chest computed tomography (CT) or diagnostic fluoroscopy guidance; 3) chest radiography; 4) admission to a tertiary medical institution or outpatient visit at a Department of Thoracic and Cardiovascular Surgery or Pulmonology at a tertiary medical institution; and 5) a diagnostic code involving lung-related disease. Lung-related diseases were selected based on Wiener et al. , and ameFor the analysis of complications among patients who underwent PTNB, we excluded patients who underwent PTNB between January 1, 2007 and December 31, 2008 in order to restrict the sample to new cases of PTNB using a 2-year pre-index period, and we also excluded those who were diagnosed with a disease related to the complication before the date of PTNB in order to identify incident cases of complications.Cases involving a complication of PTNB were defined as pneumothorax (ICD-10: J93) requiring thoracostomy or percutaneous catheter drainage, using the corresponding HIRA procedure codes on the same date as the PTNB.The covariates that were analyzed included age group , sex, insurance status , imaging guidance (CT guidance or fluoroscopy guidance), year of PTNB, comorbidities , Charlson Comorbidity Index (CCI), and history of lung surgery during the 1-year period before the PTNB date. The CCI score was calculated using the method proposed by Quan et al. . The chaBaseline characteristics were described using proportions for categorical variables. The age-and sex-specific biopsy rate per 100,000 population was calculated by dividing the number of patients who underwent PTNB each year by the number of beneficiaries of Korean national health insurance in that year and multiplying the result by 100,000. Data from the Korea National Census conducted in 2010 were used to define the standard population. Direct standardization was used to calculate the annual age- and sex-standardized biopsy rate, with 95% confidence intervals (CIs) estimated based on the Poisson distribution. The age-standardized rate was calculated for sex-specific populations, and annual trends were tested using a regression model Considering the possibility that the patients were clustered within medical institutions, the incidence rate (%) of complications was estimated using the generalized estimating equation (GEE) method, assuming an exchangeable correlation structure for analyzing the correlated data, and the 95% CI of estimates was calculated using empirical standard errors . The incAssociation between risk of severe pneumothorax and characteristics of patients and hospital were performed using GEE model. We applied a GEE to account for correlation within institutions, assuming the binomial family, a logit link function, and an exchangeable correlation structure. The odds ratios (ORs) and 95% CIs of severe pneumothorax associated with age, sex, insurance status, imaging guidance, type of medical institution, region, presence of chronic obstructive pulmonary disease, pleural effusion, lung surgery and CCI score were calculated in univariable analysis. After forward selection method was used, the multivariable models included age, sex, imaging guidance, type of medical institution, region, presence of chronic obstructive pulmonary disease, and CCI score.All statistical analyses were conducted using SAS Enterprise Guide, version 6.1 .The present study was exempted from review by the Institutional Review Board of Seoul National University Hospital (IRB number: E-1604-054-753).From the HIRA database, we identified 73,132 biopsies of 66,754 patients who met our criteria for PTNB of the lung from 2007 to 2015, and among them, 55,687 patients were included in the analysis of complications .The baseline characteristics of patients who underwent PTNB of the lung from 2007 to 2015 are shown in The crude biopsy rate was 16.7 per 100,000 population in 2007, and 24.3 per 100,000 population in 2015. The age-/sex-standardized rates were 19.6 per 100,000 population in 2007 and 22.4 per 100,000 in 2015, with a peak in 2012. The age-standardized rate in 2015 was 29.2 per 100,000 in men and 15.9 per 100,000 in women. The biopsy rate showed a significantly increasing trend (p = 0.008), both in men (p = 0.019) and women (p = 0.0039) .The incidence rate of severe pneumothorax was 2.4% (95% CI: 2.15%-2.73%) among patients who newly underwent PTNB during 2007–2015, with a breakdown by sex of 2.9% (95% CI: 2.55%-3.21%) for men, and 1.4% (95% CI: 1.14%-1.69%) for women. The incidence rate of severe pneumothorax increased by age group, ranging from 1.6% in those aged 20–49 to 2.6% in the 80+ age group. The incidence of PTNB-related severe pneumothorax was 2.5% in CT-guided PTNBs, in contrast to 2.1% in fluoroscopy-guided PTNBs. The incidence rate was lower in the Seoul metropolitan area  than in urban areas  and rural areas , and it was lower in superior general hospitals  than in general hospitals  .After adjustment, significant ORs for severe pneumothorax were found for sex, age group, the presence of COPD, region, and type of medical care institution. The adjusted OR (aOR) for severe pneumothorax was 1.84 (95% CI: 1.61–2.11) in men compared to women. Patients in their 60s, 70s, and those over 80 showed an increased risk  compared with patients <50 years. The presence of COPD , residence in an urban region , and receiving treatment at a general hospital  were linked with an increased risk of severe pneumothorax .Our study evaluated the annual rate of PTNB, the rate of severe pneumothorax as a complication, and factors related to the incidence of PTNB-related severe pneumothorax in Korea using a nationally representative database. The age-/sex-standardized rates showed an increasing trend from 2007 to 2015, both in men and women. The rate of severe pneumothorax as a complication was 2.4%. The risk factors of PTNB-related severe pneumothorax were male sex, older age, the presence of COPD, residence in urban and rural regions, and type of hospital.The increasing trend of PTNB was interpreted as resulting from increasing rates of participation in opportunistic cancer screening programs. Although screening for lung cancer was not included in the organized cancer screening program implemented by the Korean government, in recent years, most general hospitals have provided cancer screening programs. The number of patients participating in opportunistic cancer screening programs has increased, despite their relatively high cost . FurtherThe complication rate in the current study was in line with the results of previous studies. In previous studies, post-biopsy severe pneumothorax requiring intervention occurred in 1%-14.2% of patients , 26–28. Older age was identified as a risk factor for complications of PTNB. As the age of patients increased, the incidence of complications increased. This could be explained by age-related physiological changes, such as weakening of the elastic recoil of lung parenchyma. Similarly, Geraghty et al. reported that being more than 60 years old was a risk factor for pneumothorax after transthoracic needle biopsy, and the risk remained when old age was defined as 70 or older . In singThe presence of COPD increased the risk of pneumothorax by 1.27 times after adjusting for other covariates, and similar findings were reported in previous studies that identified COPD as a risk factor , 17, 32.The complication rate of severe pneumothorax was relatively low at superior general hospitals, and the association between the level of medical institution and the complication rate may be explained by differences in clinicians’ proficiency due to the case volume at larger institutions. According to a notification of the Ministry of Health and Welfare, the treatment of serious diseases that require high-tech treatment such as surgery, as well as treatments requiring the use of multiple facilities and specialized equipment, fall within the scope of superior general hospitals, and centralization among upper-level institutions was also reported for lung cancer surgery .Compared with metropolitan regions, urban regions were associated with an increased risk of PTNB-related severe pneumothorax. A similar trend was found among patients undergoing outpatient transthoracic needle biopsies in the US . Lung caHowever, the results of our study need to be interpreted cautiously. First, since the HIRA database did not have detailed information on patients’ clinical condition, the accuracy of the diagnoses could not be confirmed. To overcome this limitation, we defined complications using diagnosis and procedure codes that were essential for reimbursement. Another limitation is that we included patient-level factors and characteristics of medical institutions as risk factors for complications, however, only the COPD and pleural effusion were contained as comorbidities, and other comorbidities including emphysema, interstitial lung diseases, and congestive heart failure were not considered. Further studies with comprehensive risk factors would be desirable. Lastly, the characteristics of the target lesions  and proficiency of operating physicians or radiologists which were relevant factors in risk of pneumothorax could not be evaluated in this study due to the lack of information in the claims database. Furthermore, these information is the basis for judging the appropriateness of modality selection. Transbronchial biopsy during bronchoscopy is preferred for lesions near to bronchial tree, because it does not cross the pleura, transbronchial approach has been reported lower incidence of pneumothorax . HoweverThis is the first nationwide study to evaluate the practice patterns of PTNB in Korea and to calculate the complication rate of PTNB-related severe pneumothorax and related factors using a health insurance database that includes the entire Korean population. Considering the increasing trend of PTNB, more attention needs be paid to patients with risk factors for complications of PTNB.


Optimizing cardiac stroke volume during high-risk surgical anesthesia is of particular interest with regard to a therapeutic target to reduce the incidence of postoperative complications. However, intensive fluid management in critically ill small animals with pulmonary hypertension (PH) has been empirically performed, and thus it can be challenging. Stroke volume variation (SVV) has been used as a dynamic preload predictor of fluid responsiveness. We hypothesized that if SVV exhibited robust reliability in the setting of hemodynamically unstable condition, it would provide more precise information on fluid resuscitation to translate it into veterinary anesthesia. Thus the aim of this study was to investigate the utility of SVV measured by the electrical velocimetry (EV) method for predicting fluid responsiveness in dogs with PH.Sixteen dogs undergoing emergency abdominal surgery and diagnosed with PH secondary to myxomatous mitral valve disease (MMVD) on preoperative transthoracic echocardiogram were included. Dogs were randomly assigned to 2 groups with and without inotropic cardiac support with dobutamine. Hemodynamic measurements including stroke volume and SVV derived from the EV device were performed under general anesthesia before (baseline) and after surgery .P < 0.05) with a greater number of responders than saline infused control group (P < 0.01). Receiver operating curve analysis of SVV confirmed high positive predictive value for dogs during dobutamine infusion .In both groups, SVV elevated significantly after abdominal surgery compared with baseline. In dobutamine infused group, the SVV values decreased significantly after fluid challenge (Noninvasive EV monitoring may be useful for the prediction of fluid responsiveness in critically ill dogs with left-sided heart failure-related PH. This normalization of dynamic preload indices, which could be achieved more precisely under inotropic support, may prevent further detrimental consequence of fluid loading. Optimizing cardiac stroke volume during surgical anesthesia is of particular interest with regard to a therapeutic target to reduce the incidence of postoperative complications. Pulmonary hypertension (PH) is related to the risk for development and severity of clinically significant congestive heart failure . There iAssessment of hemodynamic and volume status can be achieved by intensive fluid management guided by a combination of cardiac output (CO) monitoring and preload indices. Pulmonary or transpulmonary thermodilution and uncalibrated arterial pressure waveform analysis can provide valuable information for understanding hemodynamically unstable conditions in experimental animals –15. HoweRecently, a new noninvasive monitoring system, without the need for external calibration, using the electrical velocimetry (EV) method has been introduced in dogs with experimental cardiac surgery –19. In areal-world” emergency surgical setting of hemodynamically unstable condition, it would provide more precise information on fluid resuscitation to translate it into veterinary anesthesia and critical care. Thus the aim of this study was to investigate the utility of EV-based SVV for predicting fluid responsiveness in dogs with PH secondary to MMVD undergoing emergency abdominal surgery. In this study, fluid challenge was performed in the presence of inotropic support with dobutamine to ameliorate the ‘false-positive’ effect of SVV elevation attributable to PH in lowering afterload . Rectal temperature was maintained at 37–38°C using a forced-air patient warmer . Anesthesia was maintained with sevoflurane (1.0–2.0%) in 60% oxygen for an adequate depth of anesthesia as described previously  [-1 min-1) or vehicle sodium chloride 0.9% w/v  at a constant infusion rate (2 ml kg-1 hour-1) [This study measured hemodynamic variables in sevoflurane-anesthetized dogs at n (MAC)]  with a c hour-1) , 39.-1 IV over 15 minutes) of a colloid solution ]. Dogs were considered fluid responsive if SVI measured immediately after completion of the fluid challenge increased by ≥10% ) for a level of significance of 0.05 and a power of 80% to be achieved .P-value of 0.05 was considered statistically significant. Statistical analyses were performed using SPSS , Prism , and SigmaPlot .All variables were compared to the baseline  and among the groups (DOB vs. Control). Data are expressed as mean ± SD, unless otherwise mentioned. Normally distributed data among groups were determined with analysis of variance (ANOVA), and non-normally distributed variables were analyzed with Kruskal-Wallis test. When statistical significance level has been reached, parametric (Bonferroni test) and non-parametric (Scheffe test) univariate comparisons were performed. Fisher’s exact test was used to compare the categorical variables. The predictive ability of a variable for fluid responsiveness was assessed using a receiver operating characteristic (ROC) curve calculated with a 95% confidence interval. For each variable, a threshold value was determined to maximize both sensitivity and specificity. A Table 1. All dogs were extubated and recovered without procedure- or therapy-related complications or death.Baseline characteristics of the 16 dogs underwent emergency abdominal surgery  are presented in Table 2. There were no statistically significant group differences between the baseline phase. Hypovolemia induced by intraoperative bleeding and resected tissue (368 ± 135 g) (Table 1) caused a significant increase of SVV as compared to baseline , the degree which was greater in the DOB group  .Hemodynamic data with and without DOB groups under different volumetric conditions are shown in P < 0.05), in which dobutamine infused group had a greater number of responders (63%) than saline infused group (25%) (P < 0.001). Before volume loading, the values of SVV were significantly higher in responders defined by an increase of SVI ≥ 10% (P = 0.035), whereas those of SVV were not (P = 0.39) .In DOB groups, SVV decreased significantly after volume expansion  . The optimal cut-off values were 15.0% for SVV during DOB infusion (90% specificity and 82% sensitivity) (Table 3). Corresponding grey zone limits of SVV were between 13.5% and 16.5%.The ROC curve analyses for SVV illustrated that fluid responsiveness for the identification of responders was predicted better with DOB infused group [AUC = 0.97 ± 0.06 (standard error), The principal finding of this study is that noninvasive SVV analyzed by the EV device is valuable for the prediction of fluid responsiveness in dogs with PH and MMVD undergoing emergency abdominal surgery. This normalization of dynamic preload indices, which could be achieved more precisely under inotropic support, may prevent further detrimental consequence of fluid loading.In this study, the EV-derived SVV is assumed to be an adequate predictor of fluid responsiveness in the setting of PH secondary to MMVD. However, it should be noted here that preload requirements for PH differ substantially based on whether afterload is normal or increased. The PH and volume loading can induce displacement of the interventricular septum toward the LV and subsequent decline of RV perfusion. In this setting, intravascular volume dehydration should be needed to avoid exacerbating bi-ventricular cardiac dysfunction because of less chance of improvement on RV contractility over a wide range of filling pressures due to flatter Starling curve of RV than the LV . This ma1 receptor stimulation, but it may also cause vasodilatation due to β2 effects with increasing doses [-1 min-1) because it restores pulmonary artery/RV coupling in a canine study [-1 min-1) should be avoided in this setting because of the risk of β2-mediated vasodilatation and hypotension in healthy dogs [-1 min-1 to maintain CO close to normal range by minimizing adverse drug effects in emergency surgical patients with variety range of systemically disease histories [In this regard, our results suggest that supplementary inotropic support improved fluid responsiveness to dogs with MMVD-related PH. Dobutamine is an inotrope that acts via βng doses . In thisne study  and imprne study . The benne study . Althougthy dogs . Sinus tthy dogs . In factistories , 11, 14.Although only a few studies have directly compared SVV with other estimates of dynamic indices , 51, ournoninvasive EV methodology in dogs under intravascular hypovolemia attributable to losses of body fluids to 'third spaces' . Furthermore, this study was carried out before and after abdominal wall closure for ethical aspects of veterinary patients’ safety. While previous studies have suggested that dynamic preload indicators such as PPV and SVV are able to predict fluid responsiveness during major abdominal surgery [This study has some limitations that may concern its readers. Currently, various functional haemodynamic variables  have been employed to estimate volume responsiveness in dogs , 53–59.  surgery , 61, the surgery . Future Although no standardized criteria exist to define hemodynamic consequences in canine high-risk surgical patients under PH secondary to MMVD, the results in this study using a noninvasive EV technique for monitoring both dynamic preload status and CO suggest the technique would be valuable for estimating fluid responsiveness.S1 Fig(TIF)Click here for additional data file.S2 Fig(a) Anesthesia monitor and electrical velocimetry (EV) device, (b) Automated anaesthesia record system, (c) Infusion pumps for controlled IV fluid administration, and (d) Positions of electrodes for noninvasive EV monitoring in the clinical setting of a dog suffering from septic shock due to ruptured gallbladder. In our institute, hemodynamically unstable small animal patients, the EV system is routinely used for monitoring of surgical anesthesia.(TIF)Click here for additional data file.


Microfluidic paper-based analytical devices (μPADs) have been suggested as alternatives for developing countries with suboptimal medical conditions because of their low diagnostic cost, high portability, and disposable characteristics. Recently, paper-based diagnostic devices enabling multi-step assays have been drawing attention, as they allow complicated tests, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which were previously only conducted in the laboratory, to be performed on-site. In addition, user convenience and price of paper-based diagnostic devices are other competitive points over other point-of-care testing (POCT) devices, which are more critical in developing countries. Fluid manipulation technologies in paper play a key role in realizing multi-step assays via μPADs, and the expansion of biochemical applications will provide developing countries with more medical benefits. Therefore, we herein aimed to investigate recent fluid manipulation technologies utilized in paper-based devices and to introduce various approaches adopting several principles to control fluids on papers. Fluid manipulation technologies are classified into passive and active methods. While passive valves are structurally simple and easy to fabricate, they are difficult to control in terms of flow at a specific spatiotemporal condition. On the contrary, active valves are more complicated and mostly require external systems, but they provide much freedom of fluid manipulation and programmable operation. Both technologies have been revolutionized in the way to compensate for their limitations, and their advances will lead to improved performance of μPADs, increasing the level of healthcare around the world. Paper-based diagnostic devices have been widely used for the past decades because of their capability to enable cost-effective point-of-care testing (POCT). In addition, their simple operation and capillary action without the need for external equipment for fluid transport have accelerated the spread of such technology. Besides these characteristics, POCT has been highlighted as an appropriate technology for developing countries with suboptimal medical conditions, as it enables cheap and quick diagnosis. Many diagnostic products, such as the dipstick test and lateral flow assay (LFA), have already been commercialized, which are of single-test type and do not require the injection of additional reagents. These single-step testing formats have been applied to conventional diagnostic applications, such as glucose, lactate, and uric acid assays . HoweverPaper-based diagnostic devices for multi-step assays can be separated into three techniques: (1) creating channels on paper ,6,7, (2)Second, the sensing technology for detecting analytes in samples is directly related to the performance of the diagnostic devices ,9,10. DeFluid manipulation technologies enable reagents and samples to be delivered to the corresponding regions in proper assay time. Fluid manipulation technology can be largely divided into passive and active valves by the presence of external inputs , and thecP is the capillary pressure, R is the radius of the capillary tube, γ is the fluid surface tension, and θ is the solid–liquid contact angle. Based on combination with Hagen–Poiseuille’s law, based on the assumptions of the fluid—laminar, incompressible, and Newtonian—the fluid in this paper follows the Lucas–Washburn equation . T. T49]. TPaper-based analytical devices fundamentally exploit capillary action to transport the fluid. Although the control of fluid by relying solely on capillary action in μPADs is so passive that it limits the flexibility of fluid manipulation, it is still the most widely used method due to its simplicity, affordability, and expandability. The chemical treatment and geometry transformation of papers can be critical parameters in terms of fluid control in μPADs. A number of methods have been used to manipulate fluid delivery, including adjustment of the paper dimensions, control of the paper permeability, switching strategies, and change of the surface chemistry of the fluidic path on the paper.Chemical-based fluid manipulation is a method of changing the wicking properties of paper by chemical treatment and can be divided into the fluid velocity control technique and the switching technique. Above all, in the aspects of fluid control techniques, a great deal of research, including a method to delay the flow rate using dissoluble material  . When thA novel technique based on the pressure valve has been introduced as an example of an active valve in the fluid manipulation section . Like a Molecular diagnostics is a technique used to analyze biological markers in the genome using gene amplification techniques, such as polymerase chain reaction (PCR). This method identifies a disease infection by extracting DNAs and RNAs containing the pathogen’s genetic information from the saliva and blood of the infected person and then amplifying them. The most popular method for molecular diagnostics is PCR, but it is limited because it requires expensive equipment with thermal cycling. However, with the development of isothermal amplification technology, DNA strands can be amplified without the thermal cycling process. The isothermal amplification method includes loop-mediated isothermal amplification (LAMP) , helicasIsothermal amplification comprises three steps: sample preparation, nucleic acid amplification, and detection. Tang et al. implemented the whole operation process of HDA within an integrated paper-based platform . After sELISA is a method of detecting and quantifying specific antigens or antibodies in biological samples, such as blood, urine, tissues, and cells. ELISA can be divided into direct ELISA, indirect ELISA, and sandwich ELISA according to the detection method. Sandwich ELISA is widely used because it shows higher specificity than the other methods. The processes of sandwich ELISA include stepwise reactions with several materials, such as the analyte, the enzyme-coupled detection antibody, and the substrate. The intermediate washing steps are also essential. Several studies have been conducted to implement ELISA in paper-based devices ,121,122,1 in Our group recently developed a paper-based ELISA system using pressure valves . The preA signal enhancement assay is a method to improve the detection signal and sensitivity. Because it requires several amplification steps , it should be accompanied by fluid manipulation techniques for application in paper-based diagnostic devices. Thus far, several studies have been done on paper-based signal enhancement assays .E. coli O157:H7 and Salmonella typhimurium. Lutz et al. performed stepwise signal enhancement through paper strips using dissolvable materials (sucrose) . As showAnother approach to enhance sensitivity is to integrate an online sample stacking, enabling the use of a less sensitive detection modality . Li et a2O2) and gluconic acid. Peroxidase then catalyzes the reaction of H2O2 with a color indicator, resulting in a color change. In this process, potassium iodide (KI) is the most commonly applied color indicator, resulting in a reaction showing brown coloration as KI is oxidized. In addition to the application based on glucose assay, colorimetric enzymatic assay in µPADs is widely used in various fields, such as pesticide assay [Colorimetric detection is the most widely used technology for paper-based analytical devices, which involves visual observation of the color change owing to the reaction of various reagents. This method enables either qualitative or quantitative analysis by taking advantage of the fact that it can be measured with the naked eye or with the help of simple visual readers. In particular, a colorimetric enzymatic assay using reactions depending on the concentration of analytes and enzyme, such as a glucose colorimetric assay, is a representative method in μPADs ,60. Glucde assay ,33, ion de assay ,103, andde assay . These µ2+ and nitrite using plastic comb binding spine (PCBS)-based valves [Renault et al. developed hollow channel paper devices for glucose and protein assays A. When td valves . AlthougIn recent years, many advances have been made in fluid handling in the field of paper microfluidics. Advances of fluid manipulation technology have led to improved performance and diverse applications of microfluidic paper-based analytical devices. In this paper, the fluid handling technologies were classified into passive and active methods according to the operation principle, which has advantages and disadvantages, respectively. Passive valves have the advantages of being able to reduce the cost and being more portable because the valve is configured in the device itself without any additional equipment. Based on the benefits, the approach is expected to evolve toward a way that can support faster response and higher integration with compact fashion. Active valves are advantageous for more accurate and programmable fluid manipulation because of the excellent responsiveness of the valves. However, since active valve methods are accompanied by additional equipment, cost-effective and straightforward modules will be desirable. In terms of practical utility and commercialization, the delicate calculation will be required between the cost increase by additional active valve modules and the expected values by reduction of reagents and performance enhancement. What is clear is that if the value of lateral flow assays—a simple, convenient, and cheap diagnosis—itself is fading away, it will face practical limitations no matter how sophisticated the active valve technology is. As such, it would be desirable to conduct technical development within the scope of the original value of paper-based platforms.The potential requirements of fluid manipulation technologies for multi-step assays in paper-based analytical devices can be summarized as follows: (1) compact form, (2) instant response, (3) repeatable use, and (4) automated operation. The development of paper-based multi-step assay technologies through fluid handling techniques can bridge the current disparity in medical advancements globally. For instance, complicated assays for many viral and infectious diseases can be performed on-site in undeveloped countries if the assays currently being performed in the laboratory are implemented in paper-based technology. Therefore, it can accelerate the time to commercialization if it (1) meets the ASSURED  criteria of the World Health Organization (WHO), which is the standard of point-of-care testing and, at the same time, (2) solves the challenges with full automation and obtains high sensitivity of paper-based diagnostic technology capable of multi-step assays. In addition, providing medical benefits to developing countries can greatly contribute to the diagnosis of diseases caused by super bacteria or viruses and can help prevent disease spread.