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download assay guidance manualAffiliations 1 National Center for Advancing Translational Sciences, National. Translational Sciences; 2004 -. Copyright andCommons Attribution-NonCommercial-ShareAlike 3.0 Unported licenseAny altered, transformed, or adapted form of theOriginally written as a guide for therapeutic project teams within a majorTopics addressed in thisMore than 100 authors from around the globe have contributed content to this freeFor more information about the Assay Guidance Manual and related trainingContents Expand All Collapse All New in Assay Guidance Manual Preface G. Sitta Sittampalam and Nathan P. Coussens. Published May 1, 2012; Last Update: March 31, 2017. Acknowledgements Considerations for Early Phase Drug Discovery Editors: Michelle Arkin, Nathan P. Coussens, Viswanath Devanaryan, Zhuyin Li, and G. Sitta Sittampalam. Early Drug Discovery and Development Guidelines: For Academic Researchers, Collaborators, and Start-up Companies Jeffrey Strovel, Sitta Sittampalam, Nathan P. Coussens, Michael Hughes, James Inglese, Andrew Kurtz, Ali Andalibi, Lavonne Patton, Chris Austin, Michael Baltezor, Michael Beckloff, Michael Weingarten, and Scott Weir. Published May 1, 2012; Last Update: July 1, 2016. Abstract Background Purpose Scope Assumptions Definitions Section 1. Discovery and Development of New Chemical Entities Section 2. Repurposing of Marketed Drugs Section 3. Development of Drug Delivery Platform Technology Section 4. Alternative NCE Strategy: Exploratory IND Section 5. Orphan Drug Designation Conclusion References Abstract Definitions Consequences of using Enzymatically Impure Enzyme Preparations. Signs of Enzymatic Contamination Solutions for Enzymatic Contamination Importance of Batch Testing Identity and Mass Purity Assay Design Factors that Affect the Likelihood of Detecting Enzyme Impurities Validating Enzymatic Purity References Basics of Enzymatic Assays for HTS Harold B. Brooks, Sandaruwan Geeganage, Steven D. Kahl, Chahrzad Montrose, Sitta Sittampalam, Michelle C.http://tortugafilms.com/adminfiles/canon-np-6050-manual.xml
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Smith, and Jeffrey R. Weidner. Published May 1, 2012; Last Update: October 1, 2012. Abstract Enzyme Assay Development Flow Chart Introduction Concept Reagents and Method Development Detection System Linearity Enzyme Reaction Progress Curve Measurement of K m and V max Determination of IC 50 for Inhibitors IC 50 Determination for SAR Optimization Experiments Assay Validation References Mechanism of Action Assays for Enzymes John Strelow, Walthere Dewe, Phillip W Iversen, Harold B Brooks, Jeffrey A Radding, James McGee, and Jeffrey Weidner. Published May 1, 2012; Last Update: October 1, 2012. Abstract Overview of MOA in Drug Discovery Types of Inhibition Performing MOA Studies References Glossary of MOA Terms Assay Development for Protein Kinase Enzymes J. Fraser Glickman. Published May 1, 2012; Last Update: October 1, 2012. Abstract Introduction Assay Development Considerations of Mechanism Assay Optimization Acknowledgements References Additional References: Receptor Binding Assays for HTS and Drug Discovery Douglas S Auld, Mark W. Farmen, Steven D. Kahl, Aidas Kriauciunas, Kevin L. McKnight, Chahrzad Montrose, and Jeffrey R. Weidner. Published May 1, 2012; Last Update: July 1, 2018. Abstract Introduction Flow Chart of Steps to Assay Development for SPA Format Flow Chart of Steps to Assay Development for Filter Format Scintillation Proximity Assays (SPA) SPA Assay Format Assay Buffer Solvent Interference Conditions Binding Parameter Filtration Assays Filter Assay Format Assay Buffer (Filter) Assay Conditions (Filter) Binding Parameters (Filter) Practical Use of Fluorescence Polarization in Competitive Receptor Binding Assays References Protease Assays Guofeng Zhang. Abstract Introduction Homogenous Assays Separation-Based Assays Triaging Assay Hits Data Analysis for Time Dependent Inhibitors and Covalent Inhibitors Conclusion References Inhibition of Protein-Protein Interactions: Non-Cellular Assay Formats Michelle R. Arkin, Marcie A.http://www.oipipleszno.pl/userfiles/canon-np6221-repair-manual.xml Glicksman, Haian Fu, Jonathan J. Havel, and Yuhong Du. Published March 18, 2012; Last Update: October 1, 2012. Abstract Overview and Introduction ELISA-type assays Mix-and-read assays Validating drug-like binding of PPI inhibitors Useful websites References Additional References Immunoassay Methods Karen L. Cox, Viswanath Devanarayan, Aidas Kriauciunas, Joseph Manetta, Chahrzad Montrose, and Sitta Sittampalam. Published May 1, 2012; Last Update: July 8, 2019. Abstract Flowchart Abbreviations Introduction and Background General Considerations for HAT Assays Biochemical HAT Assays Section Summary Cell-Based HAT Assays Miscellaneous Considerations Conclusions Acknowledgements Conflict of Interest Statement Suggesting Readings (alphabetical order) References Published May 1, 2013. Published May 1, 2013; Last Update: July 1, 2016. Abstract Introduction Conclusion References Published January 21, 2016. Abstract Flow Chart: 3D Spheroid and Microtissue Growth and Assay Development Introduction 1. In Vitro Toxicity and Drug Efficacy Testing in a 3D Spheroid Model 2. 3D Microtissue Viability Assay References Cell-Based RNAi Assay Development for HTS Scott Martin, Gene Buehler, Kok Long Ang, Farhana Feroze, Gopinath Ganji, and Yue Li. Published May 1, 2012; Last Update: May 1, 2013. Published October 1, 2012. Abstract 1. Introduction 2. Fluorescence Assays Using Membrane Potential Sensing Dyes 3. Ion Flux Assays 4. HTS Assay Considerations 5. Preparation of Cells for Automated Electrophysiology References Automated Electrophysiology Assays Birgit T. Priest, Rok Cerne, Michael J. Krambis, William A. Schmalhofer, Mark Wakulchik, Benjamin Wilenkin, and Kevin D. Burris. Published March 9, 2017. Published July 1, 2016. Published November 20, 2017. Published May 1, 2019.https://www.thebiketube.com/acros-bosch-logixx-8-instruction-manual Abstract Introduction Dyes That Selectively Penetrate Dead Cells Fluorescent DNA Binding Dyes That Penetrate Dead Cells Optional Protocol to Estimate Total Number of Cells Markers That Leak Out of the Cytoplasm of Dead Cells into Culture Medium Conclusion References Pharmacological Characterization of GPCR Agonists, Antagonists, Allosteric Modulators and Biased Ligands from HTS Hits to Lead Optimization Chi Shing Sum, Brian J. Murphy, Zhuyin Li, Tao Wang, Litao Zhang, and Mary Ellen Cvijic. Published November 1, 2019. Abstract Introduction to GPCRs as Drug Targets GPCR Signaling from a Drug Discovery Perspective High-Throughput Screening Assays for Identification and Optimization of GPCR Ligands Pharmacological Deliverables of HTS and Lead Optimization Important Pharmacological Considerations During Lead Optimization How the On- and Off-Rates of the Test Agonist (Compound Kinetics) Affect Compound Functional Activity Measurements and Thereby Impact Lead Optimization Dose-Shift Assays Dose-Shift Assays in Screening Mode Biased Signaling Conclusion Acknowledgement Keywords References Published May 1, 2012; Last Update: October 1, 2012. Abstract Experimental Design Flowchart Materials and Methods References Assay Artifacts and Interferences Editors: Michelle Arkin, Douglas Auld, Jonathan Baell, Kyle Brimacombe, Jayme L. Dahlin, Timothy L. Foley, James Inglese, and Stephen C. Kales. Compound-Mediated Assay Interferences in Homogenous Proximity Assays Nathan P. Coussens, Douglas Auld, Philippe Roby, Jarrod Walsh, Jonathan B. Baell, Stephen Kales, Kamyar Hadian, and Jayme L. Dahlin. Published February 1, 2020.http://anthonycohen.com/images/bunn-o-matic-model-vps-manual.pdf Abstract Flowchart Abbreviations Introduction to Proximity Assays Chemical Mechanisms of Assay Interference in Homogenous Proximity Assays Strategies to Identify Compound-Mediated Interference in Homogenous Proximity Assays Strategies to Mitigate Compound-Mediated Interferences in Proximity Assays Conclusions Suggested Readings (alphabetical order) Suggested Web Resources Glossary Acknowledgements Conflict of Interest Statement References Assay Interference by Chemical Reactivity Jayme L. Dahlin, Jonathan Baell, and Michael A. Walters. Published September 18, 2015. Abstract Flow Chart Abbreviations Introduction and Background Knowledge-Based Strategies to Minimize Impact of Interference Compounds Experimental-Based Strategies to Mitigate the Impact of Reactive Interference Compounds Conclusions Suggested Websites Suggested Readings (alphabetical order) References Interference with Fluorescence and Absorbance Anton Simeonov and Mindy I. Davis. Published December 7, 2015; Last Update: July 1, 2018. Abstract Flow Chart Fluorescence Interferences Absorbance Interferences Acknowledgements References Interferences with Luciferase Reporter Enzymes Douglas S. Auld and James Inglese. Published July 1, 2016; Last Update: July 1, 2018. Abstract Introduction Interferences with Reporter Enzymes Strategies to Mitigate Luciferase Inhibitor Interference References Assay Interference by Aggregation Douglas S. Auld, James Inglese, and Jayme L. Dahlin. Published July 26, 2017. Abstract Flowchart Abbreviations Introduction and Background Strategies to Mitigate Incidence of Aggregation in Biochemical Assays Strategies to Identify Aggregation Frequently Asked Questions Conclusions Suggested Web Resources Suggested Readings (alphabetical order) Acknowledgements REFERENCES Assay Validation, Operations and Quality Control Editors: Viswanath Devanaryan, Timothy L. Foley, Madhu Lal-Nag, Jeffrey R. Weidner, and Mary Jo Wildey. Basic Guidelines for Reporting Non-Clinical Data Jayme L.https://c2mag.com/wp-content/plugins/formcraft/file-upload/server/content/files/16295073b6753b---community-physical-activity-planning-a-resource-manual.pdf Dahlin, G. Sitta Sittampalam, Nathan P. Coussens, Viswanath Devanarayan, Jeffrey R. Weidner, Philip W. Iversen, Joseph V. Haas, Duane D. Bronson, O. Joseph Trask, Jr., Todd R. Wiernicki, and Steven D. Kahl. Published October 1, 2019. Last Update: July 8, 2017. Abstract Introduction Chromatography Instrument Set Up Data Handling Other Notes References Impedance-Based Technologies Kim E. Garbison, Beverly A. Heinz, Mary E. Lajiness, Jeffrey R. Weidner, and G. Sitta Sittampalam. Published May 1, 2012. Abstract Introduction Common Equipment in HTS Labs Microplates Microplate Sealing Microplate Readers Spectrophotometry pH Meters Electronic Balances Microscopes Liquid Handling Devices Suggested Websites and Resources Suggested Readings (alphabetical order) Calculations and Instrumentation used for Radioligand Binding Assays Steven D. Kahl, G. Sitta Sittampalam, and Jeffrey Weidner. Abstract Introduction Radioactive Calculations Instrumentation Uniformity Plate Color Quench Correction Abbreviations Pharmacokinetics and Drug Metabolism Editor: Xin Xu. Abstract Flow Chart of a Two-tier Approach for In Vitro and In Vivo Analysis Background In Vitro Analysis - Low Compound Requirements and Relative Moderate Capacity Example of In Vitro ADME Profiling Assays In Vivo Analysis - High Compound Requirements and Low Capacity Suggested Equipment and Resources References Glossary Glossary of Quantitative Biology Terms Viswanath Devanarayan, Barry D. Sawyer, Chahrzad Montrose, Dwayne Johnson, David P. Greenen, Sitta Sittampalam, Terry Riss, and Lisa Minor. Published April 15, 2012; Last Update: September 22, 2014. Abstract Cell Culture Terms Quantitative Biology Terms Genetics Terms Expand All Collapse All Previous Editors: Lisa Minor, PhD, Vance Lemmon, PhD, Andrew Napper, PhD, John MCommons Attribution-NonCommercial-ShareAlike 3.0 Unported licenseAny altered, transformed, or adapted form of theClin Transl Sci. 2018 Sep; 11(5):461-470. Williams KP, Scott JE. Assay Guidance Manual.agrobrasilia.com/userfiles/files/997-manual-pdf 2004 See reviews. See all. Recent Activity Clear Turn Off Turn On Assay Guidance Manual Assay Guidance Manual Your browsing activity is empty. Activity recording is turned off. Turn recording back on See more. Get the latest research information from NIH. Tribal knowledge is any unwritten, well-tested information that is not commonly known by others within an institution. Well-tested methods outlined in the manual address appropriate statistical ways to analyze assay results and accommodate minor changes to assay protocols to ensure robustness. More than 100 authors from around the world have contributed content to this free resource, which is updated quarterly and housed by the National Library of Medicine. The chapters have PubMed citations for the contributing authors. Learn more about these training opportunities. Read the journal article. Your commerce experience may be limited. Please update your browser to Internet Explorer 11 or above. When you select your country, you agree that we can place these functional cookies on your device. After that, you will need to contact Customer Service to unlock your account. Please try again or contact Customer Service. Please request another reset link. Please try again or contact Customer Service. A verified email address is required to access the full functionality of your Promega.com account. Please try again or contact Customer Service. Please try again or contact Customer Service. Please try again or contact Customer Service. Please check your network settings and try again. Please try again or contact Customer Service. The manual includes guidelines for selection, development and optimization of various in vitro and in vivo assays used for drug development. In this article, we highlight three chapters within the Assay Guidance Manual about cell viability assays, Short Tandem Repeat profiling analysis and cell-based assays for the detection of protein:protein interactions. But where do you begin.https://www.helpfulhunks.com.au/wp-content/plugins/formcraft/file-upload/server/content/files/16295074a7b338---Community-planning-event-manual.pdf Which assays should you use. How do you analyze and validate results. To help answer these questions, the NIH Chemical Genomics Center (NCGC) published an eBook called the Assay Guidance Manual (AGM). This eBook began as an internal guide within a pharmaceutical company, but now contains more than 40 chapters written by drug discovery scientists from academic, government and industrial research laboratories. The AGM eBook includes guidelines for selection, development and optimization of various in vitro and in vivo assays and how to adapt these assays for high-throughput screening. It also provides information on commercial products, potential assay interferences and statistical validation of assay performance. Promega scientists contributed three chapters in the AGM; one on cell viability assays, another on authentication of human cell-lines using Short Tandem Repeat (STR) profiling analysis, and a third on cell-based assays for the detection of protein:protein interactions. We highlight these three chapters in this article. ATP can be measured using a bioluminescent assay with reagents containing stabilized luciferase and luciferin substrate. In the presence of ATP from viable cells, luciferase uses luciferin to generate luminescence, which is detected with a luminometer. The resulting luminescent signal is proportional to the number of viable cells. This method is great for high-throughput applications due to its superior sensitivity in higher density microwell plates. Many older cell viability assays require incubation of a reagent with viable cells to convert a substrate to a colored or fluorescent product that can be detected with a plate reader with absorbance or fluorescence capability. The resulting signal is proportional to the number of viable cells, because dead cells lose the ability to convert substrate to product.http://thanhlamresort.vn/wp-content/plugins/formcraft/file-upload/server/content/files/16295075499b4a---community-pharmacy-policy-and-procedure-manual.pdf This is the basis for many of the commonly used cell viability assays, including tetrazolium reduction (such as MTT or MTS), resazurin reduction and live-cell protease activity assays. These assays are less sensitive than bioluminescent methods and are used in lower throughput applications. In this assay, an engineered luciferase and a prosubstrate (which is not a substrate of luciferase) are added directly to the culture medium. The prosubstrate can penetrate cell membranes and enter cells. However, only viable cells with active metabolism can reduce the prosubstrate into a substrate for luciferase. The substrate then exits the cell where it is used by luciferase in the detection reagent to generate a luminescent signal. The same wells can be measured repeatedly for 3 days. The main advantages of this method are that it allows simple kinetic monitoring to determine dose response using fewer plates and cells. Also, because the method does not require cell lysis, the same cells can be used in additional cell-based assays or downstream applications.ATP can be measured using a bioluminescent assay with reagents containing detergent, stabilized luciferase and luciferin substrate. The detergent lyses viable cells, releasing ATP into the medium. In the presence of ATP, luciferase uses luciferin to generate luminescence, which can be detected within 10 minutes using a luminometer (see figure below). The ATP assay is faster than other methods since it does not require incubation to convert a substrate into a colored product. The assay has excellent sensitivity and broad linearity, making it highly compatible with high-throughput assays. It is also less prone to artifacts than other methods.Live-cell protease activity can be measured using a cell-permeable fluorogenic protease substrate (GF-AFC). The substrate enters live cells where it is cleaved by live-cell protease to generate a fluorescent signal proportional to the number of viable cells (see figure below).accofire.com/ckfinder/userfiles/files/996-turbo-tiptronic-vs-manual.pdf Because this method does not lyse cells, it allows for multiplexing with many other assays in the same sample wells, including bioluminescent cell-based assays.Only viable cells with active metabolism can reduce resazurin into resorufin, which is pink and fluorescent. This method is relatively inexpensive and more sensitive than tetrazolium assays. However, fluorescence from compounds being tested may interfere with resorufin readings.Thus, color formation can be a useful marker of viable cells. However, the incubation time for this method is long (usually 4 hours). Also, the formazan product is insoluble, so a solubilizing reagent must be added prior to recording absorbance readings. There is no need to add a solubilizing reagent since the resulting formazan is soluble, making it more convenient. Since the signal gradually increases over time, a decrease in viable cells during this long incubation cannot be detected. As illustrated in the following image, assays used to detect dead cells include measuring the leakage of a component (usually an enzyme marker) from the cytoplasm into the culture medium or the penetration of an otherwise non-permeable dye into cells with a compromised membrane. The general concept is that trypan blue is excluded from live cells but penetrates dead cells with a damaged plasma membrane. The main disadvantages of this technique are: the error involved with measuring a single sample, the subjective judgement of the user to determine what is a dead cell or stained debris, inconsistency among operators, and the time and manual labor involved with measuring multiple samples. The most important and practical factors to consider when choosing a dye include: the emission wavelength, selectivity for staining DNA, cell permeability, solubility at the vendor-recommended concentration, detection sensitivity and cytotoxicity. Fluorogenic DNA dyes that readily pass through the intact cell membrane and stain the nucleus of live cells should not be used for measuring dead cells. The figure below shows the effects of three different DNA-binding dyes continuously exposed to four different cell types for 72 hours before measuring cell viability using an ATP assay. The most common marker used for this type of assay is lactate dehydrogenase. The reducing capacity of NADH can be used to reduce a variety of substrate molecules into products that are either colored, fluorescent, or luminogenic. Figure 5 illustrates the general scheme and assay chemistry used to detect LDH-release from the cytoplasm of dead cells. The reducing power of NADH is used to convert the substrate (resazurin) into the fluorogenic product (resorufin). Initiate primary and secondary management necessary for the emergency management of acute lifethreatening conditions in a timely manner. 4. In a given simulation, demonstrate the following skills, which are often required during initial assessment and treatment of patients with multiple injuries: a. Primary and secondary assessment of a patient with simulated, multiple injuries b. Establishment of a patent airway and initiation of assisted ventilations c. Orotracheal intubation on adult and infant manikins Course Ob jec ti v e s The content and skills presented in this course are designed to assist doctors in providing emergency care for trauma patients. The concept of the “golden hour” emphasizes the urgency necessary for successful treatment of injured patients and is not intended to represent a fixed time period of 60 minutes. Rather, it is the window of opportunity during which doctors can have a positive impact on the morbidity and mortality associated with injury. The ATLS course provides the essential information and skills for doctors to identify and treat life-threatening and potentially life-threatening injuries under the extreme pressures associated with the care of these patients in the fast-paced environment and anxiety of a trauma room. The ATLS course is applicable to clinicians in a variety of situations. The burden of injury is even more significant, accounting for 18 of the world’s total diseases. Motor vehicle crashes (referred to as road traffic injuries in n FIGURE 2) alone cause more than 1 million deaths annually and an estimated 20 million to 50 million significant injuries; they are the leading cause of death due to injury worldwide. Improvements in injury control efforts are having an impact in most developed countries, where trauma remains the leading cause of death in persons 1 through 44 years of age. Significantly, more than 90 of motor vehicle crashes occur in the developing world. Injury-related deaths are expected to rise dramatically by 2020, and deaths due to motor vehicle crashes are projected to increase by 80 from current rates in lowand middle-income countries.Data from Global Burden of Disease, 2004. Reproduced with permission from Injuries and Violence: The Facts. Geneva: World Health Organization Department of Injuries and Violence Prevention; 2010. Trimodal Death Distribution First described in 1982, the trimodal distribution of deaths implies that death due to injury occurs in one of three periods, or peaks. The first peak occurs within n FIGURE 1? Road traffic mortality rate, 2013. Reproduced with permission from Global Health Observatory Map Gallery. During this early period, deaths generally result from apnea due to severe brain or high spinal cord injury or rupture of the heart, aorta, or other large blood vessels. Very few of these patients can be saved because of the severity of their injuries. Only prevention can significantly reduce this peak of trauma-related deaths. The second peak occurs within minutes to several hours following injury. The golden hour of care after injury is characterized by the need for rapid assessment and resuscitation, which are the fundamental principles of Advanced Trauma Life Support. The third peak, which occurs several days to weeks after the initial injury, is most often due to sepsis and multiple organ system dysfunctions. Care provided during each of the preceding periods affects outcomes during this stage. The first and every subsequent person to care for the injured patient has a direct effect on long-term outcome. The temporal distribution of deaths reflects local advances and capabilities of trauma systems. The development of standardized trauma training, better prehospital care, and trauma centers with dedicated trauma teams and established protocols to care for injured patients has altered the picture.The black line represents the historical trimodal distribution, and the bars represent 2010 study data. Reprinted with permission from Gunst M, Ghaemmaghami V, Gruszecki A, et al. Changing epidemiology of trauma deaths leads to a bimodal distribution.His tory The delivery of trauma care in the United States before 1980 was at best inconsistent. In February 1976, tragedy occurred that changed trauma care in the “first hour” for injured patients in the United States and in much of the rest of the world. An orthopedic surgeon was piloting his plane and crashed in a rural Nebraska cornfield. The surgeon sustained serious injuries, three of his children sustained critical injuries, and one child sustained minor injuries. His wife was killed instantly. The care that he and his family subsequently received was inadequate by the day’s standards. The surgeon, recognizing how inadequate their treatment was, stated: “When I can provide better care in the field with limited resources than what my children and I received at the primary care facility, there is something wrong with the system, and the system has to be changed.” A group of private-practice surgeons and doctors in Nebraska, the Lincoln Medical Education Foundation, and the Lincoln area Mobile Heart Team Nurses, with the help of the University of Nebraska Medical Center, the Nebraska State Committee on Trauma (COT) of the American College of Surgeons (ACS), and the Southeast Nebraska Emergency Medical Services identified the need for training in advanced trauma life support. A combined educational format of lectures, lifesaving skill demonstrations, and practical laboratory experiences formed the prototype ATLS course. A new approach to providing care for individuals who suffer major life-threatening injury premiered in 1978, the year of the first ATLS course. This prototype ATLS course was field-tested in conjunction with the Southeast Nebraska Emergency Medical Services. One year later, the ACS COT, recognizing trauma as a surgical disease, enthusiastically adopted the course under the imprimatur of the College and incorporated it as an educational program. This course was based on the assumption that appropriate and timely care could significantly improve the outcome of injured patients. The original intent of the ATLS Program was to train doctors who do not manage major trauma on a daily basis, and the primary audience for the course has not changed. They received injury care, but the resources and expertise they needed were not available. This was, unfortunately, typical of the way injury care was provided in most areas of the country. The creators of ATLS had seen how the coordinated efforts of well-trained providers improved survival of the seriously injured on the battlefields of Vietnam and at inner-city hospitals. Since then, ATLS-trained providers have been instrumental in the ongoing development of trauma systems. ATLS has played a major role in bringing together a core group of providers that are trained and focused on injury care. This core group has provided the leadership and the front-line clinical care that have enabled the growth and maturation of coordinated regional trauma systems. Before the second half of the 20th century, trauma centers did not exist. Injury was thought to be unpredictable instead of something that could be anticipated and include treatment plans to care for injuries. Some large public hospitals, especially those located in areas with high rates of poverty and urban violence, began to demonstrate that focused experience and expertise—among providers as well as facilities—led to better outcomes after injury. Outside of these centers, injury care remained haphazard; it was provided by the closest facility and by practitioners who happened to be available. As a result, the quality of injury care received was largely a matter of chance.