Prescribing intravenous (IV) fluid therapy is a core skill expected of qualified doctors at the point of graduation, but medical graduates often feel ill-equipped to perform this task. This lack of preparedness contributes to treatment-related patient harm. This scoping review maps the current state of published evidence about how junior doctors prescribe IV fluid therapy and learn how to do it.We searched five electronic databases and grey literature from 1994 until June 2016 for articles describing any aspect of IV fluid prescribing practice or its education. A total of 63 articles were selected for analysis. Using the WHO Guide to Good Prescribing to categorize the extracted findings, our review focuses on prescribing IV fluids in adult generalist settings.Most articles studied IV fluid prescribing from the perspective of the doctor. Junior clinicians struggled to conceptualize IV fluid prescribing as a ‘whole task’ in authentic work settings and lacked support. Educational interventions to improve IV fluid prescribing often focused on enhancing prescriber knowledge about fluid and electrolyte balance rather than execution of the prescribing task.Our understanding of IV fluid prescribing as a holistic integrated skill is patchy, as is its performance. Current IV fluid prescribing education appears insufficient to foster safe and effective practice. For education to achieve the ultimate goal of safer prescribing in workplaces, we need a clearer understanding of how healthcare professionals prescribe IV fluids in real world practice.The online version of this article (10.1007/s40037-017-0386-5) contains supplementary material, which is available to authorized users. Novice clinicians are responsible for most prescribing of intravenous (IV) fluids in hospital settings. Ensuring they are ready to perform this skill safely is a necessary concern for educators and practitioners. Understanding how doctors prescribe IV fluids and learn how to do it will aid the preparation of learners, but the extent of evidence describing such practices is not known. This exploratory literature synthesis describes the activities doctors employ while prescribing IV fluids, summarizes the nature of educational initiatives implemented to improve fluid prescription quality and proposes topics for future educational research and development.Prescribing intravenous (IV) fluids is one of the first and most frequent tasks a newly qualified doctor is expected to do , 2. ThusWhile many authorities regard IV fluids as drugs , 6 and aPrescribing assessments alone, however, do not guarantee safe practice. Earlier research has shown that prescribingis embedded in social contexts that make the performance of apparently simple tasks complex and error-prone –17. PresUnderstanding how doctors prescribe IV fluids and learn to do so could improve their preparation for practice and the quality of prescriptions once they graduate. Since there has been no review of this topic, our aim was to identify all relevant publications, draw whatever conclusions the evidence could support, and identify topics for future educational research and development. We chose to undertake a scoping review because this methodology is exploratory in nature, more inclusive than others, and aims to map current evidence relevant to a topic.The review followed the methodological steps for scoping reviews devised by Arksey and O’Malley  as revisResearch ethics approval was not required because this was a secondary analysis of literature within the public domain and no subjects participated.The research team comprised a senior specialist trainee in renal medicine (RFRM), and three physicians , a nephrologist (APM) and endocrinologist (TD)) involved in educational research and/or clinical teaching.Healthcare professionals typically learn through practice in workplace environments. This experiential learning in clinical settings makes significant contributions to learners’ development of their skills, such as prescribing IV fluids. It is essential that the circumstances of prescribing preparation closely align with what those in practice are expected to know, do and value . We wantWhile junior doctors prescribe most IV fluids, and do so ordinarily in general medical and surgical settings, we began with a very inclusive, exploratory question to obtain as full information as possible about IV fluid prescribing by anyone in any context: ‘What is known about how health professionals prescribe intravenous fluid therapy, and how they learn to do so?’We interrogated five electronic databases, specifically: PUBMED/MEDLINE, EMBASE, CINAHL, SCOPUS, and Web of Science. The search strategy was based on search terms originally used by the NICE guideline CG174   screened all abstracts retrieved by the search and rejected those that were not relevant to the practice of IV fluid therapy or any aspect of its education. The whole research team convened at this stage to assess whether the search strategy was identifying evidence sources applicable to the topic under review. We excluded 42 articles that were only available as a conference abstract, as is customary in scoping review methods. At this stage, we identified no articles exploring fluid prescribing from the perspective of pharmacists in practice. We only, therefore, describe studies that represent doctors, nurses, or students of these professions prescribing, administering, or monitoring IV fluid therapy to adult patients in hospital settings. Two authors (RFRM and GJG) independently reviewed full-text copies of all screened papers to determine their inclusion in the analysis, based on the criteria described in Table RFRM created a data extraction sheet using Microsoft Excel , and populated it with demographic and methodological details. We used the WHO Guide to Good Prescribing ; Fig. 1)1) as a fThe Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  criteriaOf the 63 articles selected for this analysis, 45 (71%) originated from the United Kingdom, four from Canada, three each from Australia and the USA, two from New Zealand, and one each from China, Iran, Kuwait, Sudan, the Netherlands and Turkey. Forty-four (70%) articles were published after 2008, the year of publication of the first UK guidelines on IV fluid prescribing . Forty-nSources of information were direct observations of practice and questionnaires. Most participants were junior doctors or medical students. Outside normal working hours, the junior doctors were usually unfamiliar with the patients they prescribed IV fluids for –29. TheyThe widely varying indications for starting IV fluid therapy  and diffBoth informally shared rules and formal guidelines influenced IV fluid prescribing. It was common practice, for example, to calculate maintenance IV fluid requirements by adding insensible losses to the previous day’s urine output . It was Textbooks usually describe IV fluid regimens for stable, healthy patients , 49 wherThe articles contained no information about what patients are asked or told. Clinical records documented the treatment indications and regimen inconsistently , 55–57 aNeither in maintenance IV fluid therapy nor IV fluid resuscitation were there any reports of how clinicians evaluated responses to treatment. There was, however, evidence from the treatment of sepsis in a National Confidential Enquiry into Patient Outcome and Death (NCEPOD) report, where it was insufficient in up to a third of cases . Staff oDespite a perceived lack of teaching about IV fluid therapy, most junior doctors and medical students regarded their ability to prescribe IV fluid therapy as acceptable, and judged themselves aware of its potential hazards , 47, 59.Multidisciplinary teams were educated to follow protocols , 67–70, These interventions improved elements of IV fluid therapy that were performed well but had less impact on elements executed poorly , 67. BetThere have also been educational quality improvement projects targeting completion of fluid balance charts and prescriptions, particularly in out-of-hours settings, where junior doctors were poorly supervised –75. The Researchers have examined how different learning environments influence monitoring and prescribing of IV fluid therapy. Although fluid balance and observation charts were acknowledged to be essential to good prescribing , 57, 76,The types of error described in the articles broadly grouped into ordering appropriate IV fluid prescriptions too late , 55, 77,Suboptimal IV fluid therapy, characterized by either excessive or inadequate replacement of fluid volume, often related to insufficient record-keeping and was a persistent problem –53, 77. The principal finding from our review is that novice clinicians struggled to practise the ‘whole task’ of prescribing IV fluids; they neglected some important aspects and did others incorrectly. Furthermore, they integrated mutually dependent parts of the task poorly. Available evidence perhaps offers clues to explain this. Junior doctors lacked skills to identify a need for IV fluids, particularly in patients who were unknown to them; these assessments occurred outside regular working hours, so senior help was probably not readily available. In the absence of supervision, clinicians prescribed IV fluids based on shortcuts learnt ‘on the job’, rather than relying on the findings of a physical examination, relevant investigations, or following formal guidelines. An over-reliance on such prescribing strategies, however, may have resulted in doctors overlooking specific aspects of patients’ needs assessments or comorbidity, especially in people admitted acutely to busy hospital environments. Support when available from senior colleagues or experts—whose practices stemmed from their historical workplace experiences and clinical specialty exposure—was inconsistent. This perpetuated broad differences in IV fluid prescribing behaviour.Errors such as delayed treatment or misprescribed IV fluids were extreme examples of such variation in performance. The knowledge or skills of individual practitioners, though important, were not the only factors causing harm as described in national accounts of practice, such as the National Confidential Enquiry into Patient Outcome and Death report. Rather, an interplay of factors in workplace environments, including work demands, quality of documentation, interprofessional communication, and working relationships, contributed to mistakes.These findings have ramifications for safe IV fluid prescribing and its education. In light of the above findings, the optimal conditions for novice prescribers acquiring proficiency in prescribing IV fluids appear contingent upon a holistic, integrated understanding of the task, exposure to workplace practices that enable its enactment in authentic contexts, and access to supportive feedback specific to individual learner needs. Novice doctors, furthermore, must also be able to negotiate the uncertainties created by the physiological variation in the patients they encounter and attend to differences in prescribing behaviour between groups of practitioners. This ability to adapt to differing requirements in work settings is itself an intellectual feat.Educational interventions for IV fluid prescribing, in contrast, principally addressed deficiencies in the conceptual knowledge of IV fluid therapy or adherence to clinical guidelines. While factual capacities are readily amenable to assessment in educational settings, development of these attributes alone appears insufficient to foster a readiness to prescribe IV fluids safely in workplace environments. Novice doctors prescribed IV fluids repetitively—enabling rehearsal of skills we consider essential to IV fluid prescribing behaviour—but did so under limited supervision, so it is uncertain whether they possessed dispositional qualities to recognize these events as opportunities for learning, or had access to meaningful feedback on the outcomes of their actions. We believe that feedback on performance is critical in developing prescribers that can distinguish between IV fluid prescriptions that are straightforward and more challenging cases requiring effortful thought or more expedient senior help. Educators or practitioners must, therefore, begin to attend to the configuration of workplace  relationships in health teams, which will be influential to the success of future interventions to improve IV fluid prescribing education, and ultimately, patient outcomes.Our review focused on the single most commonly performed duty of newly qualified doctors, the poor performance of which can have potentially lethal consequences. Such specificity on adult intravenous fluid therapy, admittedly, limits the transferability of our findings. IV fluids are distinctive for their powerful, immediate effect on core physiology and biochemistry, as exemplified by their use in the resuscitation of critically ill patients. A prescriber cannot wholly predict in advance the effect of any drug in such a dynamic situation. Responsible prescribing, therefore, means evaluating probabilities of benefit and harm, making empirical choices, and carefully monitoring effects. IV fluids, therefore, are very similar to agents such as rapid-acting insulin and antiarrhythmic drugs. At the other extreme, prescribing anti-tuberculous therapy acts on timescales of months rather than minutes. Variables are more clearly defined, probabilities more easily calculated, and empirical outcomes evolve far more slowly. Readers should take these important differences into account and regard prescribing as a practice with many variations.The exploratory nature of the scoping review methodology allowed us to include sources with a broad mix of study type and quality not ordinarily included in other syntheses. Some research on IV fluid therapy practices or its education was only published as abstracts, which did not fulfil our eligibility criteria; this may have limited the extent to which our sources comprise an authentic representation of reality, as is typical of any literature review. We invite readers from within the health professions to judge for themselves how far the findings of this study reflect the ways fluid prescribing and its education happen in similar settings.Our decision to focus on IV fluid prescribing in adult generalist settings necessarily resulted in the exclusion of subsets of the literature that explore IV fluid therapy elsewhere, specifically paediatrics and critical care. There are notable differences between children and adults, principally related to physiological responses to fluid, and the need to prescribe as guided by weight. The practices underpinning IV fluid prescribing in children follow historical assumptions that current evidence now challenges. Similarly, in critical care settings, there is no compelling evidence demonstrating a ‘default’ IV fluid therapy for every clinical situation or determining an ‘optimal’ fluid resuscitation strategy. Our decision, however, makes the problem relevant to where novice doctors begin work and, as we have seen, do so with variable supervision. Additional research could examine how these specialist areas approach IV fluid prescribing and whether insights are transferable to prescribing for adults in non-critical settings.The strength of using the Guide to Good Prescribing was that it provided a widely accepted framework to structure the findings of our review in a meaningful way. Its focus on the decision making between an individual prescriber and patient, however, likely underplays the relevance of cultural and organizational aspects we attempted to describe. This issue reflects the nature of the literature landscape we identified which emphasized behaviours of the prescriber and limited our capacity to comment on the contributions of other health professions (including nurses and pharmacists) to the co-ordination of IV fluid therapy or the contextual factors in the everyday interactions of work.The predominance of articles from the United Kingdom likely exhibits an increased national awareness of the risks of IV fluids illuminated by national reports examining the quality of health delivery in hospital settings and an ongoing public inquiry into the deaths of children from cerebral oedema secondary to hyponatraemia following administration of intravenous hypotonic saline. Government departments responded to these events by developing guidelines through expert consensus on what good practice in IV fluid therapy looks like and addressing supposed insufficiencies in preparing health professionals for this task. We hope that identifying research areas of future interest through the scoping review process opens new avenues for investigation and practice globally into IV fluid therapy and its education.Our study found a dearth of robust evidence on how clinicians prescribe IV fluids in practice and learn how to do it. We found gaps in the IV fluid prescribing literature, chiefly how clinicians monitor, evaluate, and adapt their practices. We also noted an absence of evidence describing the extent of patient involvement in prescribing decisions. Our appreciation of the ‘whole task’ remains, therefore, incomplete, and opens up an important research agenda.Our insights into the many variables that affect the quality of IV fluid prescribing and its education imply that practitioners and educators should, first, acknowledge its inherent complexity. While the literature focused on junior doctors instead of the multidisciplinary team, it is important to recognize that several health professionals coordinate IV fluid therapy delivery, including nurses, and, to a variable extent, pharmacists. As a precursor to designing interventions to improve IV fluid prescribing performance and minimize treatment harm, it seems imperative to both evaluate how IV fluid prescribing in a real-world clinical environment happens and understand the influence of other disciplines in its delivery. Our next study will attempt to illustrate this complexity by observing instances of IV fluid therapy prescribing in everyday practice and learn how clinical teams, as a collective, reach this goal. Our research will also pay attention to the administration and monitoring of IV fluids, as these activities are fundamentally related to prescribing acts and the wider goals of patient care.Novice clinicians prescribe most IV fluid therapy, yet it is an incompletely understood and imperfectly performed task. Most IV fluid prescribing education is divorced from the messiness and complexity of real-world contexts and limited in its focus. Junior doctors, therefore, are underprepared to practise the ‘whole task’ in a contextually sensitive way. Practitioners and educators wanting to improve this situation must obtain a clearer understanding of IV fluid prescribing as an integrated, applied skill in workplace settings to inform this process.Document A: Literature Search Strategies for the remaining four academic databases reviewed


To investigate the impact of systemic diseases on the occurrence of subretinal fluid (SRF) in diabetic macular edema (DME) and prognostic factors for residual SRF following three consecutive monthly intravitreal ranibizumab.  Ninety-seven eyes from 68 patients with DME who completed 3 consecutive monthly injections of ranibizumab were enrolled. Systemic parameters mainly included chronic kidney disease (CKD), hypertension, HbA1c, and insulin dependence. Renal parameters for CKD were serum creatinine, estimated glomerular filtration rate (eGFR), and serum albumin. Ocular factors were baseline central macular thickness (CMT), severity of diabetic retinopathy (DR), and status of panretinal photocoagulation (PRP). R = 0.397, p < 0.001 after partial correlation with adjustment for age and DR severity). As for CKD, lower serum albumin, but not eGFR or serum creatinine, was associated with baseline presence of SRF . Overall, lower eGFR and lower HbA1c values, contrary to popular belief, predicted the presence of residual SRF following intravitreal injections .  Chronic kidney disease had significant correlation with baseline SRF ( Tight sugar control and poorer baseline kidney function may slow the resorption of SRF after anti-VEGF injections in patients with DME in the short term. However, if left untreated, persistent subretinal fluid may be detrimental to photoreceptors and retinal pigmented epithelium (RPE) [Diabetic macular edema has been one of the leading causes of visual impairment worldwide . The patum (RPE) , 8. Mechum (RPE) , and disum (RPE) , 11. Othum (RPE) . A few sum (RPE) , 14. Diaum (RPE) . The purμm or more. Macular edema of other causes such as uveitis and retinal vein occlusion was excluded. Eyes that underwent previous intraocular surgery or intravitreal injection of any drug within 3 months prior to the inclusion were also excluded. This research adhered to the tenets of the Declaration of Helsinki, and Institutional Review Board (IRB) approval was obtained from the IRB of Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation.Ninety-seven eyes from 68 patients were retrospectively enrolled according to medical records from October 2012 to July 2016 in Taipei Tzu Chi Hospital. All eyes with diabetic macular edema had completed three consecutive monthly intravitreal injections of ranibizumab with follow-up at one month after each injection. Inclusion criteria were macular edema evidenced by initial CMT value of 300 The presence of subretinal fluid was defined as an anatomical separation between neurosensory retina and retinal pigmented epithelium under the fovea. Both subretinal fluid and central macular thickness was documented by OCT  at baseline and 3 months after ranibizumab injections. The presence of SRF in each eye was deemed by different surgeons. The interobserver agreement for SRF was validated in the previous study . As to bU test was used to examine the difference in renal parameters in chronic kidney disease. Discrete variables were analyzed by chi-square test or Fisher's exact test. Spearman's correlation was used to evaluate the correlation of baseline subretinal fluid to clinical parameters, and partial correlations were also performed to adjust for age and DR severity. To take into account of correlations between 2 eyes from 1 subject, binary logistic regression models with generalized estimating equation were used to determine the prognostic factors for presence of residual subretinal fluid after 3 injections. SPSS for Windows  was used for statistical analysis. A p value less than 0.05 was considered statistically significant.For continuous variables such as age, CMT, and HbA1c levels, GLM/ANOVA analysis was performed for comparison among groups with different kidney functions, and nonparametric Mann–Whitney μm. Data were further categorized according to different kidney functions  had PDR and 33 (48.5%) had chronic kidney disease. Subretinal fluid was presented in 25 patients (36.8%) before treatment. Overall, mean age was 61.5 ± 9.0 years, average glycosylated hemoglobin level was 7.2 ± 1.3%, and mean baseline central macular thickness was 419 ± 102 unctions .R = 0.397, p < 0.001). The correlations of clinical parameters to baseline subretinal fluid were presented in p = 0.028, Fisher's exact test). In PDR, baseline subretinal fluid was associated with the timing of PRP . PRP naïve eyes had the highest percentage of baseline subretinal fluid before treatment, 61.9%, compared with 42.9% and 20.6% in PRP within 6 months and beyond 6 months, respectively.Before treatment, PRP naïve PDR had a greater proportion of subretinal fluid than eyes with NPDR (p = 0.017) and higher eGFR (p = 0.043), while serum creatinine was not significantly different between presence and absence of SRF in CKD (p = 0.13)  .Subretinal fluid resolved in all cases after ranibizumab treatment except 5 eyes (5.2%). Lower baseline HbA1c and lower eGFR stages were more likely to result in residual SRF at 3 months. Age, insulin dependence, baseline CMT, and status of PRP did not affect final SRF status .Our data suggested that chronic kidney disease played a role in the occurrence of subretinal fluid in diabetic macular edema. Presence of subretinal fluid before treatment had good correlations with lower eGFR stages and lower albumin levels. To date, the reason why subretinal fluid only appears in a small portion of diabetic macular edema has remained unclear. High glycosylated hemoglobin levels and systemic hypertension have been linked to the breakdown of blood retinal barrier that makes fluid tend to shift into the subretinal space , 18. HigIn chronic kidney disease, serum albumin was a sensitive marker for the presence of subretinal fluid. Hypoalbuminemia indicated severe loss of albumin as seen in cases with nephrotic syndrome. In contrast to VEGF that causes vascular hyperpermeability and breakdown of blood retinal barrier, decrease in serum albumin may lower the intravascular osmotic pressure and increase hydrostatic pressure that leads to fluid retention in the third space, including subretinal space . Basicalμm and improved HbA1c at 36 months (p = 0.04).Opposite to the general belief, we observed that lower baseline HbA1c levels were more likely to result in residual SRF following 3 monthly intravitreal ranibizumab. While no such conclusion has been drawn from the current studies, there are some possible reasons to explain. Similar to early worsening effect mentioned in the previous literature , 27, thiOn a molecular basis, the application of Starling's rule may also provide us some insights to understand how fluid can shift across between two spaces with pressure gradient created by osmotic and hydrostatic pressure . In the There are certainly limitations in our study. First, this is a retrospective short-term study with inherent selection bias and a small sample size. The small number of residual SRF may limit the power of statistical analysis to yield a robust conclusion. We found that residual SRF after treatment occupied only approximately 5.2% of a total of 97 eyes, or 14.3% of 35 eyes with SRF before treatment. This probably reflects that three ranibizumab injections are quite effective in the resolution of subretinal fluid and macular edema in most of the cases. Second, we analyzed the spot values of clinical parameters for baseline, for example, the estimated glomerular filtration rates and serum albumin levels, which may fluctuate over time. We did not obtain values at 3 months to see if these changes could have significant impact on the result. Despite these limitations, this study is of clinical significance because we highlighted some important points from systemic perspectives. Moreover, this study raises concern as to whether tight sugar control really exerts greater benefits for those with diabetic macular edema and subretinal fluid under the treatment of anti-VEGF agents. Further research should be done to clarify these mechanisms.In conclusion, systemic diseases have significant correlations with the presence of subretinal fluid in diabetic macular edema. Chronic kidney diseases are related to the presence of initial and residual SRF after three monthly ranibizumab injections. Tight control of glycosylated hemoglobin, however, is more likely to have residual fluid possibly due to effect of osmotic balance where serum glucose plays a major role. Thus, the impact of systemic conditions should be taken into account for evaluation of response to anti-VEGF treatment.Supplementary Tables. Table S1. Baseline demographics in eyes with diabetic macular edema. Table S2. Correlating factors for central macular thickness after ranibizumab.


According to the scintigraphic results, the diagnosis of hepatic hydrothorax and tuberculosis was confirmed easily and the treatments of the patients were managed rapidly.Large symptomatic pleural effusions can be seen in cirrhotic patients with ascites. In this report, two cirrhotic patients with ascites and large pleural effusions were evaluated. To determine the cause of pleural effusion and to understand whether there was a communication of fluid between peritoneal and pleural cavities,  A large effusion in a patient with cirrhotic ascites is described as hepatic hydrothorax which is a complication of cirrhosis and found in about 4% to 6% of cirrhotic patients in the absence of primary pulmonary, pleural, or cardiac disease -4. UsualAlthough the exact mechanism of hepatic hydrothorax is controversial, it is probably due to the ascites fluid that is transported directly into the pleural space. A number of different mechanisms have been proposed to explain the development of hepatic hydrothorax, including: hypoalbuminemia and a decrease in colloid osmotic pressure; leakage of plasma from hypertensive azygos veins; lymphatic leakage from thoracic duct; passage of fluid from peritoneal cavity to the pleural space via lymphatic channels in the diaphragm; and passage of fluid into the pleural space directly via defects in the diaphragm -7.Among the different mechanisms proposed, it is mostly accepted that peritoneal fluid flows into the pleural space directly, along a pressure gradient through congenital or acquired fenestrations connecting these two spaces. The mechanism can be explained in such a way that the fenestrations in the tendinous portion of the diaphragm are covered with pleuroperitoneum and they are raised into blisters as intra abdominal pressure rises by ascites. Eventually blisters rupture and microscopic defects may appear. Positive intra abdominal pressure coupled with negative intrathoracic pressure causes a direct flow of fluid from abdominal cavity into the pleura. A pleural effusion develops if the flow exceeds the absorptive capacity of pleura .In clinical practice, presence of communication between pleural and peritoneal cavities may be successfully demonstrated by peritoneal scintigraphy. In this technique, firstly, radiolabeled tracer should be simply introduced into the ascitic fluid by paracentesis. Later, scintigraphic images of abdomino-thoracic region are obtained by gamma camera. Using this technique, demonstration of the presence of a communication between abdomen and thoracic cavities can be quickly confirmed -11.99mTc sulfur colloid peritoneal scintigraphy was used to detect whether there is such a passage of ascites fluid from the peritoneal cavity to the pleural space via the diaphragmatic defects.With this study we presented two cases of pleural effusion in which Two  cirrhotic patients with ascites and pleural effusions were evaluated to determine the cause of fluid accumulation and were searched whether there was a communication between peritoneal and pleural cavities. In addition to clinical evidence of ascites and pleural effusion, patients were also confirmed by paracentesis and thoracentesis of fluids. Protein content of ascitic and pleural fluids was used to estimate whether the fluid was due to chronic liver disease.99mTc sulfur colloid instilled the peritoneal cavity under sterile conditions. Patients were then asked to turn frequently from side to side for proper mixing of radiopharmaceutical into the ascitic fluid. Approximately 10 - 15 min after injection of the tracer, each patient was positioned supine under a large field-of-view single-head gamma camera , fitted with a low-energy general purpose collimator. An anterior abdominal image was acquired in a 256 x 256 byte matrix for 400 kcounts to confirm the presence of tracer in the peritoneal cavity. Thereafter, images of upper abdomen and chest in anterior and posterior positions were obtained at 30 min intervals for 6 h. Twenty-four-hour images were also acquired in the second patient (Case 2) since no activity was noticed in the chest region until 6 h. Markers placed at the costal margins and xyphoid to show anatomic orientation. Scintigraphic images were evaluated by two experienced nuclear medicine physicians for the presence or absence of radiotracer in pleural regions.After aspiration of ascitic fluid, approximately 37 MBq of 99mTc sulfur colloid into the peritoneal space, rapid accumulation of radioactivity in the right pleural effusion was noted  chest radiograph of the patient demonstrated a large pleural effusion evident in the right hemithorax . Prompt as noted , suggestas noted .99mTc sulfur colloid into the peritoneal space, no accumulation of radioactivity in lung region was observed up to 24 h  and 99mTc albumin colloid have been used [99mTc sulfur colloid because of the ready availability and low cost. This technique is very easy to perform. It is safe, cheap, minimally invasive, and it has low radiation dose to the patient. For its effectiveness and meaningful results, peritoneal scintigraphy has already been used to diagnose of hydrothorax not only in patients with cirrhosis but also in patients on continuous ambulatory peritoneal dialysis (CAPD) [In clinical evaluation, practice of ordering every conceivable body fluid test on every pleural fluid specimen is expensive and seems more confusing than helpful, especially when unexpectedly abnormal results, such as left sided pleural effusions in cirrhotic patients, are encountered. A rapid and simple model method for establishing whether there is any communication between two cavities is to inject a radioactive tracer into the peritoneal cavity. Other diagnostic modalities of transdiaphragmatic fenestration have included intraperitoneal instillation of air or contrast agents, MRI and surgical exploration by thoracoscopy -19. In oeen used . We useds (CAPD) .Initial demonstration of the presence of this communication may play a vital role in patient management because it may not only stop further efforts to investigate other causes but also quickly start treatment. In our first patient, the pleural effusion was shown to be due to the passage of ascitic fluid across the diaphragm via the diaphragmatic defects or the lymphatic channels. In addition to clinical features and biochemical findings of ascites and pleural fluid, with the scintigraphic demonstration of transdiaphragmatic passage, patient was confirmed to have hepatic hydrothorax. In the second patient, there was not any communication seen between ascitic and pleural fluids. Lack of communication between two fluids led to separate investigations of thorax and abdomen. Invasive procedures, such as broncoscopy and laparascopic peritoneal biopsy procedures, were performed to further explore the patient. Only after the laparoscopic demonstration of tuberculous granulomas, certain diagnosis was established and anti-tuberculosis treatment was started.99mTc sulfur colloid peritoneal scintigraphy to identify abnormal transdiaphragmatic communication between thoracic and abdominal cavities may play a critical role in the management of ascites with pleural effusion; it may help to start treatment quickly and, furthermore, may save healthcare resources.In conclusion, use of


In cancer patients, circulating tumour-derived DNA (ctDNA) levels imperfectly reflect disease burden apparent on medical imaging. Further evaluation of ctDNA levels over time is needed to better understand the correlation with tumour growth and therapeutic response. We describe ctDNA kinetics within an orthotopic, immunocompetent preclinical rabbit model of local-regionally advanced head and neck squamous cell carcinoma (HNSCC). Monitoring primary tumour and metastatic lymph node volume by computed tomography (CT), we observed a correlation between ctDNA levels and tumour burden. We found that ctDNA detection could precede evidence of tumour on CT. Sensitivity and specificity of ctDNA detection in this model was 90.2% (95% C.I.: 76.9–97.3%) and 85.7% (95% C.I.: 67.3–96.0%), respectively. Rapid tumour growth followed by auto-necrosis and tumour volume contraction produced a spike in ctDNA levels, suggesting that viable tumour cells may be required for sustained ctDNA release. Following surgical resection, both ctDNA and total plasma DNA were correlated with recurrent tumour volume. Our results reveal the complex kinetic behaviour of ctDNA and total plasma DNA upon tumour growth or surgery. This pre-clinical model could be useful for future studies focused on elucidating mechanisms of ctDNA release into the circulation from primary and metastatic sites. Cell-free plasma DNA is composed of predominantly short (~140–200 bp) DNA fragments, and is derived from both normal and malignant tissues. Circulating tumour-derived DNA (ctDNA) is shed into the bloodstream following cell death, and levels are related to tumour stage and prognosis6. Potential clinical applications of ctDNA analysis include screening, prognostication, monitoring of disease burden, evaluation of treatment response, minimal residual disease detection, and early detection of recurrence7.Recent refinements to diagnostic practices and prognostic classifiers of cancer have exploited tumour-specific molecular characteristics. Unlike tissue-based biomarkers that often necessitate invasive procedures and cannot easily be serially assessed, blood-based biomarkers have the potential to reflect tumour burden and provide insight into longitudinal tumour behaviour in a convenient and timely manner. Cancer is characterized by somatic genetic aberrations9, ovarian10, breast10, colon4 and prostate cancer11. In nasopharyngeal cancer patients for example, plasma Epstein–Barr virus DNA levels fall precipitously following surgery or radiotherapy and frequently rise again months prior to clinical detection of recurrence14. Moreover, a recent analysis of CA 15-3 (a plasma protein biomarker), circulating tumour cells, and ctDNA in patients with advanced breast cancer revealed that ctDNA served as the most rapid indicator of treatment response15. A more complete understanding of ctDNA kinetics has nonetheless been hampered by inherent challenges with serial phlebotomy of cancer patients receiving therapy. Pre-clinical animal models can provide an alternative system for assessing ctDNA kinetics17; however, to date these models have relied on subcutaneous tumour injections into immunocompromised hosts. Such non-physiologic conditions potentially limit the generalizability of findings from these studies. Thus, there is a need to develop new preclinical models for the analysis of ctDNA kinetics.ctDNA levels change over time and in response to treatment. An increase of ctDNA level has frequently been found to correlate with higher overall disease burden in multiple cancer types, such as melanoma18. Head and neck squamous cell carcinoma (HNSCC) classically has carried a poor overall prognosis; however, patients with human papillomavirus (HPV)-related oropharyngeal cancer, which has increased in incidence over the past 3 decades19, are now recognized to have higher survival rates than their HPV-negative counterparts21. Still, outcomes for HPV-related HNSCC patients remain heterogeneous, so further risk stratification strategies are warranted. Recent studies have demonstrated that HPV DNA is detectable in the majority of patients prior to treatment and declines during and after treatment24. From this prior work, ctDNA shows great promise as a biomarker that could improve the precision of risk stratification and identify patients with residual or recurrent disease after treatment.Head and neck cancer is the sixth most common cancer type worldwide25, and plasma HPV DNA kinetics in animal models have not been reported. Here, we examine ctDNA kinetics in an orthotopic, immunocompetent rabbit model of papillomavirus-associated squamous cell carcinoma. The VX2 tumour derived from the domestic rabbit is associated with the cottontail rabbit papillomavirus (CRPV), which like HPV participates in papillomatosis and carcinogenesis of infected squamous epithelium27. The transplantable VX2 carcinoma can be serially propagated in rabbits and used to model various cancer types including HNSCC31. After being injected into the buccal mucosa, VX2 tumour cells metastasize to regional lymph nodes, thus mimicking the natural history of HPV-related HNSCC in humans. This study provides insights into the mechanisms of ctDNA release into the circulation from primary and metastatic sites over time and in response to surgical therapy.There are currently few suitable pre-clinical animal models for HPV-related HNSCC31. We first confirmed that following tumour injection, tumours would form and that metastases could be detected within regional lymph nodes  and a maximum volume of 17,945.43 mm3 (Rabbit 3) prior to surgery. The mandibularis caudalis lymph node that was closest in proximity to the primary tumour also exhibited growth in tumour-bearing animals (Rabbits 1–8). As expected, the lymph node did not exhibit growth in the animal lacking a primary tumour (Rabbit 9).We analyzed the pattern of tumour growth and lymphatic spread of the VX2 HNSCC animal model. Tumour and lymph node volumes were determined at each time point for all nine VX2 tumour injected rabbits Fig. . Eight rAlthough the primary tumour in each animal initially appeared as a solid mass on CT, most tumours eventually developed central hypodensity, indicating auto-necrosis. For example, the primary tumour in Rabbit 3 was detectable by day 11 and became auto-necrotic from day 18 onwards Fig. . SimilarTerminal surgery was eventually performed on all rabbits, at which point, the tumours and metastatic lymph nodes were resected to confirm malignancy in conjunction with histological analyses Fig. . StaininE6 open reading frame (ORF) was selected for candidate assays  the tumour volume contracted after an initial phase of rapid growth Fig. . ctDNA lIn order to assess ctDNA kinetics following primary tumour resection, once tumours formed we performed survival surgery on Rabbits 6, 7 and 8. In each case, ctDNA was readily detectable immediately prior to surgery Fig. . We obseFollowing surgery, ctDNA levels fell with a half-life of 23–52 min Fig. . ctDNA bTo investigate the weaker correlation between ctDNA and tumour volume observed in the post-operative compared to the pre-operative setting, we performed histopathologic analysis of resected tissues at end point Fig. . For RabE6 ORF, which is similar to published ctDNA detection methods for HPV-associated malignancies24.Detection of ctDNA has emerged as a promising strategy for tracking tumour burden and assessing response to treatment, yet few studies have been performed to characterize the kinetics of ctDNA release in tumour-bearing subjects with local-regional disease. For this purpose, we have adopted the VX2 rabbit model of papillomavirus-associated HNSCC, which phenotypically mimics the human disease counterpart in terms of its metastatic lymph node spread in an immunocompetent host. We developed a robust qPCR assay with high sensitivity and specificity for papillomavirus DNA derived from VX2 tumour cells. This assay is based on detection of the viral Using this model, we observed a correlation between tumour volume and ctDNA originating from the injected VX2 cells. ctDNA detection displayed high sensitivity and specificity for tumour growth detected by CT. In the pre-operative setting, the strongest correlation was observed between primary tumour volume and ctDNA, whereas little-to-no correlation was observed between tumour volume and total plasma DNA, which includes DNA from normal non-malignant cells such as peripheral blood leukocytes. Although we also observed correlation between ctDNA levels and lymph node growth, this correlation was numerically weaker and could partially be explained by coincident tumour growth at the primary site.One rabbit displayed intermittently detectable ctDNA despite lack of any detectable tumour growth during the observation period. It is possible that a tumour could have arisen in this animal with additional observation time or that a small tumour could have been present below the detection threshold of CT. We also speculate that the ctDNA signal could have emanated from the injected cells that may have died slowly, leading to continuous release of low levels of ctDNA into the bloodstream. This observation, if confirmed in future studies, could have implications for settings of tumour cell dormancy or other scenarios involving slow cellular turnover where the utility of ctDNA analysis has not been firmly established.This model can produce tumours that grow rapidly and undergo auto-necrosis. As tumours grew, we found that the rise in ctDNA levels closely resembled that of the necrotic portion of the tumour. However, in one instance, when auto-necrosis became so profound that total tumour size contracted, ctDNA levels plummeted. This case study suggests that the release of ctDNA from necrotic or necrosing tumour is time-dependent and may require continual tumour growth to fuel ctDNA release. These findings, if confirmed in larger studies, could have important implications for the use of ctDNA to monitor response to treatment, as many cancer therapies cause tumour necrosis and would thus be expected to produce a temporary spike in ctDNA levels. Our results should be interpreted with caution, however, given the small number of animals included in this study and the fact that human tumours generally display much slower growth trajectories which might affect ctDNA kinetics. The relative contribution of necrosis, apoptosis, and other modes of cell death to ctDNA levels and kinetics could be explored further with this model in future studies.In the post-operative setting, both ctDNA and total plasma DNA were weakly correlated with tumour volume. We speculate that this could be attributed to inflammatory changes in the operative bed that interfere with accurate measurement of recurrent tumour while producing DNA release from non-cancerous tissues into the circulation. These findings point to the difficulties with interpretation of post-operative imaging and the potential value of ctDNA analysis for distinguishing swelling/inflammation from recurrent disease.33, so upfront genotyping of the tumour may be necessary, or else a strategy must be employed that can simultaneously detect DNA from multiple HPV subtypes while maintaining exquisite sensitivity and specificity. Third, we note that the CRPV genome is amplified ~30-fold in VX2 cells, which exceeds the median number of integrants of HPV genomes in human cancer34. Thus, an E6 qPCR assay may be less sensitive for detection of ctDNA in the majority of patients with HPV-driven cancer. With further technological advances, however, we envision that HPV-based ctDNA detection will one day become routine in clinical practice.There may be challenges in translating results of our work into the clinical realm. First, as a proportion of total blood volume the 3 mL blood draw from rabbits utilized in our study is equivalent to ~70 mL blood taken from a human subject, which would not be practical for serial assessments. Second, multiple HPV subtypes cause human cancers, including HPV-16, -18, -31, -33, -35, and othersIn conclusion, this VX2 orthotopic model of papillomavirus-related HNSCC has provided new insights into the release of cell-free DNA into the circulation from primary tumours, metastatic lymph nodes, and normal tissues. Further characterization of ctDNA kinetics in animal models and in patient samples is warranted in order to determine its clinical suitability as a biomarker for HNSCC.6/mL) into the quadriceps of New Zealand white rabbits, and tumours were harvested after approximately 3 weeks. Harvested tumours were stored in liquid nitrogen prior to tumour induction. Prior to tumour induction in rabbits, the tumour pieces were thawed and cut using a sterile scalpel and subsequently placed onto a 70 μm cell strainer sitting on a 50 mL tube (Beckton Dickinson). A syringe plunger was used to mince the cells and ~500 μL HBSS was used to suspend the cells in the strainer to reach the concentration of 5 × 106 cells/mL.VX2 tumours were propagated by injecting 500 μL of suspended VX2 cells (5 × 106/mL) VX2 cell suspension into the cheek muscles (buccinator). Tumour development and lymph node metastases were monitored using computed tomography (CT) twice a week. The experimental protocol was approved by the Animal Care Committee (ACC) of the University Health Network, and all experiments were performed in accordance with the guidelines and regulations of the ACC.Experiments were performed using male New Zealand white rabbits weighing 2.5–3.0 kg . Male rabbits were injected with 300 μL of a high-density . Under anesthesia, rabbits were placed in ventral recumbency on the scanning bed and injected intravenously with 10 mL Omnipaque  at 0.3 mg/kg administered over one minute following 2 mL 0.2% (2 mg/mL) heparin sodium salt. MicroCT scans were acquired at 80kvp and 50 mA, image reconstruction using traditional filter back projection with graphic processing unit (GPU) improving image reconstruction speed, image resolution 0.154 mm × 0.154 mm pixel size and slice thickness in 0.154 mm.All CT-based image analysis was performed using Microview  and a custom in-house program written using MATLAB . The mean and standard deviation of the voxel signal distribution within each VOI was calculated.Volumes of tumours and the mandibularis caudalis lymph node on the ipsilateral side in each subject were manually contoured using Mimics 16.0 (x64), including the total tumour or lymph node volume, the hypodense non-ehnancing (non-viable) tumour or lymph node volume, and enhancing (viable) tumour or lymph node volume.For all rabbits in the study, 3 mL of blood was collected into EDTA tubes (Beckton Dickinson) at serial time points following tumour induction. Blood samples were collected 5–10 minutes prior to microCT imaging. The length of time that the rabbits were monitored was dictated by the size of the respective tumours and the health status of those rabbits in accordance with the University Health Network Animal Care Committee policies. Within 1 hour of blood draw, plasma was separated from the blood cell pellet by centrifugation at 2500 x g for 10 minutes. A second spin at 16,100 x g for 10 minutes was performed on the separated plasma, and the supernatant was then aliquoted in DNA/RNA LoBind microcentrifuge tubes (Eppendorf) and stored at −80 °C.LINE-1 repeat element  and a standard curve constructed from purified genomic DNA from VX2 cells. Quadruplicate qPCR reactions were carried out in 10 microliters each, consisting of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) or PowerUP SYBR Green Mastermix (Life Technologies) and 250 nM oligos. Of the 22 µL purified plasma DNA, 4 µL of a 1:10 dilution was used as input into each qPCR replicate reaction. Forty cycles of two-step thermocycling and SYBR Green detection were performed using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad).Total plasma DNA was purified from all available separated plasma (1–2 mL per time point) using the QIAamp Circulating Nucleic Acid Kit (Qiagen) without the use of carrier RNA. DNA was eluted into 100 µL AVE buffer and then concentrated using Agencourt AMPure beads (Beckman Coulter) into 22 µL TE buffer  for downstream quantitative PCR (qPCR) analysis. Total plasma DNA concentration was determined by qPCR using an assay detecting the rabbit E6 ORF were selected for PCR primer design using Primer3 (http://primer3plus.com/). Default parameters were used with the exception of maximum product size, which was set at 90 bp. The selected assay amplified a 58 bp fragment within E6 . A standard curve was constructed from purified genomic DNA from VX2 cells. Quadruplicate qPCR reactions were carried out in 10 microliters each, consisting of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) or PowerUP SYBR Green Mastermix (Life Technologies) and 250 nM oligos. Of the 22 µL purified plasma DNA, 4 µL was used as input into each qPCR replicate reaction.The concentration of VX2-derived DNA within plasma DNA was determined by qPCR. Sequences specific for the Kappapapillomavirus 2 (CRPV) 3 was chosen in order to ensure that the tumour resection would not affect critical structures and vital functions of the rabbits. The primary tumour was dissected and sent for pathology and immunostaining analysis.Surgery was performed at day 16 after tumour induction. Tumour volume of 1500 mmResected tissues were fixed in formalin, embedded in paraffin blocks, cut at the thickness of 8 μm and stained with hematoxylin and eosin (H&E) and pan-cytokeratin (AE1/AE3) using adjacent slides. All histopathology images were analyzed using ImageScope after scanning.Statistical tests and determinations of 95% confidence intervals were performed in GraphPad Prism and R. Correlation was determined using the Pearson test. For calculating sensitivity and specificity, a true positive result was defined as detectable ctDNA when tumour was visible on CT at the same or subsequent time point, a false negative result was defined as undetectable ctDNA when tumour was visible on CT at the same or subsequent time point, a false positive result was defined as detectable ctDNA in pre-injected rabbit plasma or in Rabbit 7 follow surgery (due to lack of tumour recurrence), and a true negative result was defined as undetectable ctDNA in pre-injected rabbit plasma or in Rabbit 7 following surgery. All error bars represent standard deviations. P-values of <0.05 were considered significant. All P-values were two-sided. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


The aim of this study was to investigate the challenges and feasibility of pre-clinical quantitative imaging and mouse-specific dosimetry of 111In-labelled HER2-targeting monoclonal antibodies (mAbs) (range 5.58–8.52 MBq). Sequential SPECT imaging up to 197 h post-injection was performed using the Albira SPECT/PET/CT pre-clinical scanner. Mice were culled for quantitative analysis of biodistribution studies. The tumour activity, mass and percentage of injected activity per gram of tissue (%IA/g) were calculated at the final scan time point and compared to the values determined from the biodistribution data. Delivered 111In-labelled mAbs tumour absorbed doses were calculated using mouse-specific convolution dosimetry, and absorbed doses for 90Y-labelled mAbs were extrapolated under the assumptions of equivalent injected activities, biological half-lives and uptake distributions as for 111In.Attenuation, scatter and partial volume effects were studied using phantom experiments, and an activity calibration curve was obtained for varying sphere sizes. Six SK-OV-3-tumour bearing mice were injected with r = 0.999, P < 0.0001) and the tumour mass calculations , whilst no correlation was found for the %IA/g . Median tumour-absorbed doses per injected activity of 52 cGy/MBq (range 36–69 cGy/MBq) and 649 cGy/MBq (range 441–950 cGy/MBq) were delivered by 111In-labelled mAbs and extrapolated for 90Y-labelled mAbs, respectively.For the sphere sizes investigated (volume 0.03–1.17 ml), the calibration factor varied by a factor of 3.7, whilst for the range of tumour masses in the mice (41–232 mg), the calibration factor changed by a factor of 2.5. Comparisons between the mice imaging and the biodistribution results showed a statistically significant correlation for the tumour activity (This study demonstrates the need for multidisciplinary efforts to standardise imaging and dosimetry protocols in pre-clinical imaging. Accurate image quantification can improve the calculation of the activity, %IA/g and absorbed dose. Diagnostic imaging could be used to estimate the injected activities required for therapeutic studies, potentially reducing the number of animals used. Molecular imaging enables minimally-invasive visualisation of molecular and cellular biological processes in living organisms. It plays an important role in cancer drug development and in monitoring disease progression and tumour response to therapeutic interventions –3. AnimaAbsolute image quantification is essential to evaluate imaging biomarkers and to accurately determine the distribution of the uptake of novel radiotracers to evaluate their toxicity and efficacy profile in small animals prior to use in human studies. It is also necessary for dosimetry calculations and therefore has the potential to improve our understanding of the biological mechanisms of radiation-induced cell damage , 6 and t111In-labelled monoclonal antibodies (mAbs) targeting HER2-positive tumours. In particular, the influence of tumour size on quantification accuracy was investigated.The aim of this study was to explore the challenges and potential role of quantitative pre-clinical SPECT imaging as an alternative for biodistribution studies. Methods used in the clinical setting were applied to a pre-clinical study to investigate image quantification and mouse-specific dosimetry of 111In (ca 42 MBq)  in acetate buffer (pH = 4). All reactions were performed as described previously [The HER2-targeting mAbs used in this study were the commercially available trastuzumab  and ICR12, developed at The Institute of Cancer Research, London , 19. Theeviously , 21. The6/mouse)  suspended in 30% Matrigel  diluted in HBSS . Tumours were allowed to grow for 3–4 weeks and the radioimmunoconjugates, diluted in saline, were injected via the tail vein   Act 1986 following local ethical review and the United Kingdom National Cancer Research Institute Guidelines for Animal Welfare in Cancer Research . Six fem2 mixture  and placed on the scanner bed. A 1% isoflurane gas mixture was maintained during acquisition. The imaging study was performed using the Albira SPECT/PET/CT tri-modal pre-clinical scanner (Bruker), which allows fully registered anatomical and functional imaging [2 CsI(Na) crystal and a position sensitive photomultiplier tube. All acquisitions were performed with a single-pinhole collimator and transverse and axial FOVs of 80 mm and 60 mm, respectively. An energy window width of 20% was placed on the lower gamma-ray emission of 111In at 171 keV. The scans were reconstructed using default parameters, the ordered subsets expectation maximization (OSEM) method with two iterations and five subsets into a 110 × 110 matrix with voxel size of 0.75 mm. The images were not corrected for attenuation, scatter or partial volume effects, as these are not provided by the manufacturer. The six mice were imaged at 4, 24, 48, 72 and 96 h post-injection (pi), and two mice were additionally imaged at 168 and 197 h post-injection. A CT  and a 15 min SPECT acquisition were performed at each time point.Mice were anesthetised by inhalation of a 2% isoflurane/O imaging , 23. The111In (0.1 HCl) and attached to a 30.4 ml cylindrical phantom  to simulate a subcutaneous tumour in the flank of a mouse. The phantom was placed on a moveable platform adjustable to sub-millimetre precision and scanned in ten positions: five axial positions in the centre and five positions 8 mm off-centre from the FOV trans-axially. The experiment was performed with the cylindrical phantom filled with air and water. The mean counts in the sphere were compared for both cases to assess the effects of attenuation and scatter within the mice.In-built attenuation and scatter corrections are not available in the Albira system. Therefore, a simple phantom experiment was used to investigate their combined effect. A 2.3 mm-radius sphere was filled with 32.6 MBq of 111In (0.1 M HCl) to investigate partial volume effects and to calibrate the SPECT images. Each sphere was attached to a 30.4 ml cylindrical phantom  filled with 0.1 M HCl solution to provide a realistic scatter and attenuation medium.To quantify the level of activity in the in vivo SPECT scans, phantom studies were performed to convert from measured counts to units of activity (MBq). Three spherical phantoms with volumes of 0.03, 0.12 and 1.17 ml were filled with activity concentrations in the range of 7.7–10.2 MBq/ml of Each phantom was scanned using a 15-min acquisition and reconstructed with the same parameters as for the in vivo study.http://www.vtk.org) [S-value kernel. The SPECT scans were co-registered using a rigid mutual information algorithm, and these were used to obtain the time-activity curves and to calculate the cumulated activity at the voxel level. A trapezoidal or exponential extrapolation method was used for the intermediate phases, depending on whether the activity between the two time points increased or decreased respectively. For the uptake phase, it was assumed that the activity at the time of administration was zero and linearly increased to the first scan time. For the last phase, exponential decay with physical half-life was assumed from the last scan point to infinity to avoid any bias introduced by registration errors and redistribution of uptake at the voxel level. Voxel- S value kernels were generated with an application developed using the EGS++ class library within the general purpose EGSnrc Monte Carlo (MC) code [111In and 90Y with 0.75 mm voxels in a soft-tissue homogeneous medium were generated. Simulations were carried out without variance reduction techniques and with 107 histories to maintain the statistical uncertainty below 1% at central and nearest neighbouring voxels. The decay spectra used for the simulations were obtained from the RADTABS software [Image-based convolution dosimetry was performed using in-house software , developvtk.org) . Three-dMC) code , 27, preMC) code . Kernelssoftware .3 and the tumour activity using the calibration curve obtained from the phantom experiment. The tumour uptake, determined as the percentage injected activity per gram of tissue (%IA/g), was then calculated from the activity and mass of the tumour at the final scan time point. The tumour absorbed doses delivered by 111In-labelled mAbs and those that would have been delivered by 90Y-labelled mAbs were extrapolated to demonstrate the feasibility of pre-therapy-image-based treatment planning for therapeutic studies. The assumptions of equivalent injected activities, biological half-lives and uptake distributions for 90Y-labelled mAbs as for the 111In-labelled mAbs were made.Due to insufficient contrast between the tumour and the surrounding muscle tissue on the CT images, tumours were outlined in the SPECT images using a thresholding method using the PMOD software (PMOD Technologies). The threshold was varied until the tumour edge in the SPECT scan matched the visible tumour edge in the CT image. The thresholded final SPECT images were used to calculate the tumour masses assuming a density of 1 g/cmThe mice were euthanized by cervical dislocation immediately following the final scan: 96 h for four mice and 197 h for the remaining two mice. The tumours were dissected and weighed, and the radioactivity was measured in a WIZARD2 automatic gamma counter . Tumour uptake (%IA/g), activity and mass were calculated. Comparisons between the three radiotracers were beyond the scope of this study.The activity, mass and %IA/g in the tumour calculated from the last SPECT scan were compared with those obtained from the biodistribution study, which is considered to be the gold standard measurement.P values below 0.05 were considered significant.Median and range were used to describe continuous variables. Pearson’s correlation coefficients and regression analysis were used to quantify and identify relations between variables. Two-sided 99mTc, 124I and 131I has previously been reported [111In.The radiolabelling of ICR12 with reported –32, and For the attenuation/scatter phantom experiment, the mean counts in the sphere were on average 3% lower in the presence of the water-filled cylinder than with the air-filled cylinder.Figure 111In-labelled antibodies was 52 cGy/MBq (range 36–69 cGy/MBq) and extrapolated for 90Y was 649 cGy/MBq (range 441–950 cGy/MBq)  was obtained from the biodistribution data, with a median activity of 0.093 MBq (range 0.015–0.335 MBq) and a median %IA/g of 48% (range 33–66%).r = 0.999, P < 0.0001) and mass . When activity and mass were compounded together into the uptake calculation, no relationship between imaging and biodistribution uptake values was observed , with a median difference of –30% (range –46 to 14%)  Fig. .Fig. 4Tuy-intercept of the linear regression line.The relationship between the %IA/g and mass differences between imaging and biodistribution is shown in Fig. 111In-labelled mAbs targeting HER2-positive tumours. However, a correlation was not found between uptake as calculated from the imaging and the biodistribution data. Finucane et al assessed the quantification accuracy of 111In pre-clinical imaging and concluded that imaging could replace some dissection studies for the assessment of radiotracer biodistribution in mouse models [This study investigated the challenges associated with pre-clinical imaging and mouse-specific dosimetry and compared the mass, activity and %IA/g in the tumour obtained from the imaging and the ex vivo biodistribution data. A statistically significant correlation was observed between the tumour activity and mass as calculated from the biodistribution and imaging data at 96 and 197 h following injection of e models . Their c111In was 2.1 (±0.4) mm. The investigation of the combined effects of attenuation and scatter showed only a 3% error on image quantification, which is small compared with the limitations of the spatial resolution and partial volume effects. Attenuation and scatter will play an important role for larger size rodents and low energy emitting radionuclides. For example, Hwang et al found that the measured concentration of activity in a volume of interest in the centre of a rat-sized water-filled cylinder was reduced by up to 50% for 125I and up to 25% for 99mTc [Accurate calculation of subcutaneous xenograft tumour volume is a key element of pre-clinical studies, as it is used as a metric to assess tumour growth and to quantify response to therapy, as well as for image quantification and dosimetry. Our results showed that for the range of tumours observed in this mouse cohort, the calibration factor to convert from counts to activity changed by a factor of 2.5, which can potentially lead to significant errors in the quantification of tumour activity, uptake and absorbed dose. These differences are likely due to partial volume effects, as the median tumour diameter observed in this study was 5.4 mm (range 4.3–7.6 mm), and the measured average axial spatial resolution for S values for organ and tumour-absorbed dose calculations. Several authors have studied the impact of various voxel-based mouse dosimetric reference models and found large variations in S-values due to the variability of the anatomical features [Tumour and organ masses can also have a significant impact on tabulated features –37. Our features , 39, whiThe dosimetry methodology used in this study showed the feasibility of treatment planning in pre-clinical studies, where absorbed doses can be extrapolated for any radionuclide from a given uptake distribution. This could ease experimental planning for therapeutic radiotracers by informing the level of injected activity required to deliver a given absorbed dose, in particular for theragnostic agents, with the potential to reduce the number of animals used.This study demonstrates the need for multidisciplinary efforts to standardise imaging and dosimetry protocols in pre-clinical imaging before replacing ex vivo biodistribution studies. Accurate image quantification can have a large impact on improving the calculation of mass, activity, uptake and absorbed doses and therefore has the potential to improve treatment response studies and the comparison of novel radiotracers.


The MEK1/2 inhibitor, Selumetinib  was used as a tool compound. Firstly, we established an acute treatment protocol and demonstrated this protocol could differentiate phenotypic and pharmacodynamic responses to Selumetinib (0–3uM). We then used the protocol to evaluate Selumetinib response in tumours from 23 colon cancer patients. These studies revealed that the agent inhibited pERK1/2 phosphorylation in all tumours, caused a significant decrease in proliferation in 5/23 (22%) tumours, and that KRAS/BRAF mutant tumours were particularly sensitive to the anti-proliferative effects of the agent. These data are consistent with data from clinical trials of Selumetinib, suggesting that acute treatment of small tumour biopsies is worthy of further exploration as a pre-clinical model to evaluate colorectal cancer response to novel therapies.Although an array of new therapeutics has emerged for the treatment of colorectal cancer, their use is significantly impacted by variability in patient response. Better pre-clinical models could substantially improve efficacy as it may allow stratification of patients into the correct treatment regime. Here we explore acute,  Standard chemotherapy is associated with severe side effects and provides marginal benefit to the majority of patients2. Hence, there is an overwhelming need for novel, more targeted therapies that would impact on disease progression and patient outcomes.Colon cancer remains a major cause of morbidity and mortality worldwide with one million diagnoses annually and at best, 60% survival3. Ideally, models should conserve the heterogeneity observed in patient tumours with respect to architecture, cell types  and tumour microenvironment. Traditional preclinical models of colorectal cancer, including tumour cell lines, genetically modified animals and cell line derived tumour xenografts, failed to capture this heterogeneity resulting in poor mapping to clinical outcomes5. Organoids derived from human tumours have recently gained traction as a representative model for colorectal cancer8. However, organoids do not recapitulate the heterogenous architecture of human cancers or model tumour-stromal interactions. Furthermore, the growth selection pressures applied during organoid generation has the potential to introduce bias. Another approach that is considered more representative is the use of patient derived xenografts (PDX), where a small fragment from a patient tumour biopsy is implanted either subcutaneously or orthotopically into an immunodeficient mouse10. However, the timescales and costs involved in this process are very significant. Development of alternative, innovative approaches for identifying tumours that will respond to specific agents is now essential if new therapies are to be targeted to the correct patient population.A major impediment to progress in the development and use of targeted therapies for colorectal cancer is the lack of good pre-clinical models that can accurately predict the efficacy of new drugs and determine the patient population most likely to benefitEx-vivo culture of intact tumour biopsies is potentially an extremely attractive system for analysing patient response to therapeutics as tumour architecture is retained, spatial relationships between tumour and stroma preserved, and the unique genetic and epigenetic modifications in each individual tumour incorporated13. Furthermore, biopsies from individual patients can be rapidly investigated. Supporting the validity of this model system, Vaira et al. demonstrated that vibrotome cut tumour sections grown in ex vivo culture are stable over time and exhibit the expected signalling response to PI3K inhibitors14. Furthermore, Rubio et al.15 demonstrated that the ex vivo response of fine needle pancreatic cancer biopsies to anti-cancer agents predicted the effects of the agents in vivo15.ex vivo treatment of fresh, colorectal tumour biopsies as a method to determine patient sensitivity to new therapies. The RAS/RAF-MEK1/2-ERK1/2 signalling cascade is deregulated in approximately 50% of colorectal tumours, which drives tumour progression through constitutive ERK1/2 activity18. The most common cause for this deregulation is mutation in RAS/RAF family members, with mutant KRAS  and BRAF (12% of cases) being the most prevalent19 Since MEK1/2 lies directly downstream of these mutations and uniquely phosphorylates ERK1/2, the MEK1/2 kinases have been identified as extremely attractive targets for therapeutic intervention.Here we used the MEK1/2 inhibitor, Selumetinib  as a model therapeutic to test in vitro in cell line studies and in vivo, in mouse xenograft models22. The agent has also demonstrated a manageable toxicity profile and evidence of disease stabilisation in Phase I and II clinical trials27. However, although promising, these studies have revealed considerable variability in response, with many tumours showing resistance27. Previous research aimed at stratifying patient sensitivity to the agent have primarily utilised human tumour cell lines, grown in vitro or as xenografts in nude mice. Not only have these approaches been poor at predicting outcomes in cancer patients but, they have also given inconsistent results with regards to the association between ras/raf mutations, pathway activation and drug sensitivity29. Clearly, alternative approaches are required for determining tumour sensitivity to this agent.Selumetinib is a potent, non-ATP competitive, small molecule inhibitor of MEK which has demonstrated significant anti-tumour activity against colorectal cancer ex-vivo treatment protocol and demonstrated that target inhibition and phenotypic responses to Selumetinib could be quantified using this protocol. We also demonstrated a link between KRAS/BRAF mutational status and response to low dose Selumetinib, providing proof of principle that this approach can successfully identify genotype-phenotype association. We conclude that this methodology has the potential to identify patients that may benefit from treatment with MEK inhibitors and potentially other therapeutics and as such, warrants further clinical investigation.Here we developed an acute 30. Selumetinib (AstraZeneca) was dissolved in DMSO (10 mM stock) and cells treated as specified.Colon cancer cell lines RKO, HRT118, and HCT116 were obtained from ATCC/ECACC and maintained as described2) were provided by a pathologist at the time of resection. Informed consent was obtained from all patients, full ethical approval was in place  and all procedures were performed in accordance with the relevant regulations. Biopsies were immediately placed in culturing media  and transferred to the lab (within 1 hour). Following two PBS washes, tumours were placed in a petri dish of culturing medium and dissected into 1–2 mm2 fragments. Fragments were washed again with fresh media then placed in wells of a 96 well plate immersed in culturing medium containing 10% FCS and either 0, 0.1 or 3 µM Selumetinib. All samples had a final DMSO concentration of 0.03%. Two tumour fragments were treated per condition: one for immunohistochemical analysis and one for protein extraction. Samples were incubated at 37 °C for the times specified.Small colorectal tumour biopsies (1–2 cm31.Immunocytochemistry and immunohistochemistry were carried out using standard protocols with antibodies against Ki-67 (1:100 DAKO) and active caspase-3  and fluorescently tagged secondary antibodies (Jackson Labs)31. The percentage of epithelial cells (defined by DAPI staining) positive for either Ki-67 or active caspase-3 was determined for five (immunohistochemistry) or ten (immunocytochemistry) random fields of view. In both cases, at least 190 cells per slide were analysed.Images were acquired as described31.Whole cell lysates were prepared from cell pellets and frozen tumour samples (with the aid a QIAGEN TissueLyser LT) using 1x Lysis Buffer as per the manufacturer’s instructions . Western blot analysis was performed on 30 µg of protein using antibodies to phosphorylated p44/42 MAPK (p-ERK1/2) , p44/42 MAPK (ERK1/2)  and β-actin , as previously describedImmunoblots were quantified using ImageJ software. Relative pERK1/2 intensity was determined by subtracting background intensity from pERK1/2 band intensity then normalising this value using native ERK1/2 intensity and β-actin.KRAS codon 12 and 13 mutations and BRAFV600E were investigated.Mutational analysis was performed by AstraZeneca using a Roche Cobas system. t-test unpaired with equal variance. For immunohistochemistry, P values were derived by comparing the data from the five fields of view from the untreated sample to the five fields of view from the Selumetinib treated sample. Pearsons correlation coefficient (r2) was used to examine the relationship between fold decrease in proliferation in response to 0.1 µM Selumetinib compared to 3 µM Selumetinib, for the 5 patients that showed a significant response only at the 0.1 µM dose (Subset 2).P values throughout were calculated using a two-tailed Student’s All data generated during this study, and all methods and materials utilised, are available upon request.ex vivo treatment protocol could be used as a means of determining colorectal tumour sensitivity to Selumetinib. Firstly, we investigated the use of a number of methods that reportedly allow ex vivo growth of intestinal mucosa. However, we found that for all protocols tested, prolonged time in culture (>24 h) induced a significant increase in apoptosis and decrease in proliferation in colorectal tumour biopsies (Supplementary Table acute (less than 6 h) drug exposure24.We set out to determine whether an BRAF mutant) and HRT18 (KRAS mutant) that are resistant and HCT116 (KRAS mutant) that is sensitive32. The drug concentrations used in these studies were in keeping with those reported in patient plasma after Selumetinib treatment23. Figure To test this principle, we firstly examined the pERK1/2 and phenotypic response to Selumetinib in colorectal cancer cell lines that have known sensitivity differences when exposed to the agent for prolonged periods; RKO  acted as a baseline control. A summary of clinical, molecular and TNM staging data for the patient cohort is outlined in Table ex vivo exposure to Selumetinib caused a significant, dose-dependent decrease in relative ERK1/2 activity in all colorectal tumour biopsies  and apoptotic (caspase-3) responses to Selumetinib could be detected using acute drug treatment of small tumour biopsies. For each tissue section, the percentage of positive cells in five fields of view  was quantified. The average of these five fields was then calculated and used to determine the fold decrease in proliferation/increase in apoptosis (compared to 0 µM) for each tumour at both doses Fig. . To deteex vivo exposure to Selumetinib significantly (p < 0.05) inhibited proliferation in 11/23 (47.8%) tumours  between response at 0.1 µM and that at 3 µM. On analysis of apoptotic effects we found that in 3/23 tumours there was a dose-dependent increase in apoptosis within an hour of Selumetinib treatment. However, in contrast to the anti-proliferative effect, this only reached significance at the higher dose (3 µM) of the agent  and Selumetinib response .34. Therefore, we next determined whether our model could be used to explore genotype, phenotype relationships. Firstly, our panel of tumours were evaluated for the most common KRAS (codon 12 and 13) and BRAF (V600E) mutations. Mutations in PIK3CA, which are reported to increase resistance to MEK inhibition, were also evaluated19 (Table KRAS (codon 12 and 13), two BRAF(V600E) and 2 PIK3CA mutants were identified, including one tumour with a BRAF and PIK3CA mutation  or when mutations were considered individually (i.e KRAS codon 12/13 and BRAFV600E)  mutant tumours  Fig. . There w0E) Fig. . Upon Seurs Fig. . Howeverurs Fig. . Furtherurs Fig. . In factons Fig. . In contons Fig. .Figure 5KRAS mutations have increased sensitivity to the anti-proliferative effects of Selumetinib. They also provide proof of principle that this model system can detect specific anti-proliferative effects and may be used to establish associations between genetic biomarkers and phenotypic responses to drug treatment.Collectively, these data suggest that tumours with ex-vivo treatment of small, tumour biopsies as a novel method for identifying colorectal tumour sensitivity to specific therapeutics. The MEK inhibitor, Selumetinib, was used to test the efficacy of the approach.Targeted therapy, based on the unique biological characteristics of each individual tumour, is revolutionizing treatment options for cancer patients. However, to realise the full clinical potential of this approach, better tools are required for identifying biologically discreet subpopulations of patients, so that they can be stratified to correct therapeutic regimes. Here we explored the use of acute ex-vivo treatment protocol we established that: 1) 3 µM of Selumetinib inhibits pERK1/2 phosphorylation in all tumours and the effect is independent of whether or not tumours harbour a KRAS or BRAF mutation. 2) Low dose Selumetinib induced a significant reduction in Ki-67 staining in 5/23 (22%) tumours. 3) the phenotypic responses to the agent were independent of the magnitude of target inhibition, and 4) KRAS mutant tumours were particularly sensitive to the anti-proliferative effects of Selumetinib. These findings are consistent with those of a phase I clinical study where it was shown that 5/20 biopsies of human tumours demonstrated a 2 fold reduction in Ki-67 staining after 7 days Selumetinib treatment, that all biopsies demonstrated a reduction in pERK1/2 staining, that wild-type and KRAS/BRAF mutants showed similar levels of target inhibition and that mutant tumours were more sensitive to the growth inhibitory effects of the agent23. Using xenografts of patient derived metastatic colorectal cancer grown in mice, Migliardi et al., also found that Selumetinib inhibited pERK1/2 activity in all cancers and caused disease stabilisation in 27%29. These similarities suggest that our ex vivo model system may have the potential to reveal clinical responses to MEK inhibitors. It would now be extremely interesting to undertake clinical trials alongside ex vivo treatment in order to fully assess this approach.Using the short term 34. One possibility for the discrepancy is the dose of the agent utilised. We found that the relationship between genotype and phenotype was particularly evident at 0.1 µM Selumetinib, which is a tenth of the maximum tolerated dose in humans and is a lower dose than is generally used in in vitro studies. Since resistance to MEK inhibitors in RAS/RAF mutated tumours has been attributed to negative feedback mechanisms, it is possible that at this lower dose, MEK inhibitors show more specificity for their target and are less likely to activate these feedback mechanisms. Our ex-vivo methodology would be an ideal platform to interrogate such adaptive signalling further.Our data indicate that mutations in the MAPK/ERK pathway are associated with an enhanced anti-proliferative response to low dose Selumetinib. This result was extremely encouraging with regards to the model system as it suggested the phenotypic effects we observed at low doses of the agent are valid, and that this approach has the potential to identify genotype-phenotype relationships. However, these data are in contrast to a number of studies in the field, which demonstrate the phenotypic response to Selumetinib is independent of the mutational status of this pathwayex vivo methodology. However, the protocol we established has the potential to reveal information regarding response to other agents. Indeed, we have used this protocol to examine tumour response to low doses of the chemopreventative/therapeutic agent, aspirin. Our cell line studies had demonstrated that aspirin causes an increase in levels of the NF-κB suppressor, COMMD135. Using the described ex vivo protocol with patient tumour biopsies, we could show that aspirin also caused increased COMMD1 in 3/4 colorectal tumours, again potentially identifying patients that would benefit for aspirin therapy36. Together, these data suggest that this approach has broad application.In this study we used a MEK inhibitor as a model therapeutic to test our ex vivo, these generally involve tumour manipulation (for example by sectioning or xenograft growth) prior to ex vivo culture15. The advantage of our method is that it does not require pre-manipulation, enabling the use of very fresh, extremely small tumour biopsies. As we have shown drug response (Selumetinib and aspirin) can be quantified after acute treatment using this ex vivo culture protocol, this methodology could be ideal as a fast, cost effective means of identifying patients that would benefit from Selumetinib or other therapies following surgical resection of primary tumour. Since this protocol does not allow long term growth of tumours, it could be argued that patients who respond with delayed kinetics may be overlooked. The question of whether the kinetics of the response influences the phenotypic outcome is interesting and should be addressed in clinical trials undertaken alongside ex vivo treatment.Although other groups have published protocols for assessing tumour response to therapeutics ex vivo treatment of small biopsies of human colorectal tumours has considerable potential as means to reveal patient specific responses to MEK inhibitors and other therapeutic agents and to identify markers of response. We conclude that this approach warrants further investigation.In summary, we show that acute Supporting data


To examine if methylene blue (MB) can counteract or prevent protamine (P)cardiovascular effects.The protocol included five heparinized pig groups: Group Sham -without anydrug; Group MB - MB 3 mg/kg infusion; Group P - protamine; Group P/MB - MBafter protamine; Group MB/P - MB before protamine. Nitric oxide levels wereobtained by the nitric oxide/ozone chemiluminescence method, performed usingthe Nitric Oxide Analizer 280i . Malondialdehydeplasma levels were estimated using the thiobarbiturate technique.1) Groups Sham and MB presented unchanged parameters; 2) Group P - a)Intravenous protamine infusion caused mean arterial pressure decrease andrecovery trend after 25-30 minutes, b) Cardiac output decreased and remainedstable until the end of protamine injection, and c) Sustained systemicvascular resistance increased until the end of protamine injection; 3)Methylene blue infusion after protamine (Group P/MB) - a) Marked meanarterial pressure decreased after protamine, but recovery after methyleneblue injection, b) Cardiac output decreased after protamine infusion,recovering after methylene blue infusion, and c) Sustained systemic vascularresistance increased after protamine infusion and methylene blue injections;4) Methylene blue infusion before protamine (Group MB/P) - a) Mean arterialpressure decrease was less severe with rapid recovery, b) After methyleneblue, there was a progressive cardiac output increase up to protamineinjection, when cardiac output decreased, and c) Sustained systemic vascularresistance decreased after protamine, followed by immediate Sustainedsystemic vascular resistance increase; 5) Plasma nitrite/nitrate andmalondialdehyde values did not differ among the experimental groups.Reviewing these experimental results and our clinical experience, we suggestmethylene blue safely prevents and treats hemodynamic protaminecomplications, from the endothelium function point of view. Thesecond in vitro study suggested that pulmonary circulation isextensively involved in the protamine-mediated effects on endothelialfunction. The thirdstudy, carried out in anesthetized dogs, reported the methylene blue (MB) and nitricoxide (NO) synthase blockers neutralization of the protamine vasodilatoryeffects The fourth studyproposed that protamine also causes endothelium-dependent vasodilation in heartmicrovessels and conductance arteries by different mechanisms, includinghyperpolarization.Reviewing those experimental results and our clinical experience, we suggest MB as anovel approach to prevent and treat hemodynamic complications caused by the use ofprotamine after cardiopulmonary bypass.Clinical and experimental observations prove that heparinneutralizing doses ofprotamine increase pulmonary artery pressures and decrease systemic blood pressure.Protamine also increases myocardial oxygen consumption, cardiac output (CO), andheart rate, and decreases systemic vascular resistance. These cardiovascular effectshave clinical consequences that have justified studies in this area. Protamineadverse reactions usually can be classified into three different categories, namely:systemic hypotension, anaphylactoid reactions, and catastrophic pulmonaryvasoconstriction. The precise mechanism that explains protamine-mediated systemichypotension is unknown. Four experimental protocols performed at Mayo Clinic studied the intrinsic mechanism of protamine vasodilation. Thefirst study reported In the absence of prospective clinical trials and cumulative clinical evidence, basedon the literature case reports, the present study was carried out to examine if MBcan counteract or prevent protamine cardiovascular effects.The protocol included five heparinized pig groups: Group Sham - without any drug;Group MB - MB 3 mg/kg infusion; Group P - protamine; Group P/MB - MB afterprotamine; Group MB/P - MB before protamine. NO plasma levels were measured ineach of the experimental groups. The procedures and handling of the animals werereviewed and approved by the Institutional Animal Care review board (Reg142/2006).®,Cristália Produtos Químicos Ltda., SP, Brazil) andtiletamine/zolazepam . Maintenance was achieved by total intravenous anesthesia using sufentanil and propofol  delivered by syringe infusion pump . Pancuronium bromide  was used as a muscle relaxant. Tracheostomy was performed onall animals immediately after induction of anesthesia. Volemia maintenance wasachieved with intravenous infusion of sodium chloride 0.9% (5 mL/kg/h). ASwan-Ganz CCOmbo CCO/SvO2 744HF75 catheter was placed in the right jugular vein and into the lumen of the mainpulmonary artery. The left carotid artery was simultaneously catheterized. Meanarterial pressure (MAP), pulmonary arterial pressure (PAP), pulmonary capillarypressure (PCP) and central venous pressure (CVP) were recorded by the MP System100 A . Cardiac output (CO), systemic vascularresistance (SVR) and pulmonary vascular resistance (PVR) were obtained by theVigilance System . After instrumentation, aperiod of 20 minutes was allowed for anesthesia stabilization. After that,hemodynamic parameters and clinical conditions were recorded for 15 minutes.Female Dalland pigs (22-26 kg) were induced to anesthesia with intramuscularadministration of midazolam  and analysis of variance (Twoway ANOVA) and Bonferroni post-test. TheNitrite/Nitrate (NOx) and malondialdehyde (MDA) results were analyzed usingpaired T-test . Valuesare considered to be statistically significant at P=0.05)  .P=0.3786) (In the MB infusion before protamine group (Group MB/P), the drop in MAP was lesssevere, with rapid recovery. In MB infusion after protamine group (Group P/MB),marked MAP decrease after protamine was observed, with recovery after MBinjection. The curves of P, P/MB and MB/P groups were not different and theeffect was not significant (=0.3786) .P>0.05). The statistical significance was borderline and thesoftware pointed out that a larger number of animals would improve the data. InGroup P, CO decreased and remained stable until the end of the protamineinjection  between groups P and MB/P at 30 minutesbetween groups P and Sham at 35 and 40 minutes  between group Pversus P/MB and P versus MB/P at 40minutes  after protamine injection (15 minutes),followed by immediate decrease (P<0.01) between Group P versus P/MB and Pversus MB/P at 15 minutes  binds to an unidentified endothelial cell receptor that mediates theconversion of L-arginine to NO. NO released abluminally activates soluble guanylatecyclase in the vascular smooth muscle to induce cyclic GMP (cGMP)-mediatedrelaxation (vasodilation). This results in a decrease in peripheral vascularresistance and hypotension. NO released luminally would promote thrombolysis andinhibit platelet adhesion in the blood vessel. Against this hypothesis, it is important to report thatCastresana et al., using pigvascular smooth muscle cell culture experiments, showed that protamine does notalter the responses of the intracellular second messengers, cGMP and cAMP, to thevasodilators sodium nitroprusside, atrial natriuretic peptide, isoproterenol, andforskolin. Those results do not support the hypothesis that protamine sensitizesvascular smooth muscle cells to the NO/endothelium-derived relaxing factor.Favorable to the NO/endothelium-dependent mechanism, MB has been successfully usedto reverse protamine vasoplegic reactions in humans-20.The precise mechanism that explains protamine-mediated systemic hypotension isunknown. However, it is suggested that protamine decreases peripheral vascularresistance rather than depressing myocardial function. Favorable tothis hypothesis is the use of inhaled NO to control pulmonaryhypertension-23.For the endothelium-dependent vasodilatation in the pulmonary artery, a proposedmechanism suggests that protamine sulfate binds to an unidentified endothelial cellreceptor to induce NO production from the amino acid L-arginine. NO then diffuses tothe underlying vascular smooth muscle to induce relaxation (vasodilatation). NOreleased into the lumen would promote thombrolysis and inhibit platelet adhesion inthe blood vessel. The conversion of L-arginine to NO can be inhibited byNG-monomethyl-L-arginine (L-NMMA), a methylated form of L-arginine. In addition,differently from the systemic circulation, heparin can inhibit the ability ofprotamine to induce NO, presumably by preventing the binding of protamine toreceptorPlasma NOx and indirect free radical activity estimated by measuring MDA levelssurprisingly did not show differences. Probably, the experimental time (40 minutes)was not enough for the laboratory techniques to detect the possible stimulus for NOrelease and free radical (lipidperoxidation) activity.In conclusion, based on the results described, the following can be stated: 1)Individual MB infusion did not change MAP, CO and SVR values whereas MB injectedbefore protamine attenuated the hypotension with rapid recovery and when injectedafter protamine, it reversed the marked hypotension; 2) CO decreased after protamineinfusion, recovering after post-MB infusion, and MB infusion before protamine causeda progressive CO increase, followed by attenuation at the time of protamineinjection; and 3) There was sustained SVR increase until the end of protamineinjection, decreased SVR after protamine, followed by immediate SVR increase when MBwas injected before protamine, and sustained SVR increase after protamine and postMB injections. Reviewing these experimental results and our clinical experience, wesuggest that MB prevents and treats hemodynamic protamine complications.Study limitations. It is mandatory to emphasize that the animals were not undercardiopulmonary bypass that causes systemic inflammatory reaction, since thisreaction should exacerbate the hemodynamic effects of protamine. Other relevantlimitation is the number of animals used for each group (n=6), except for borderlinestatistical significances, the statistical software pointed out that a larger numberof animals would improve the data.