Gout [1 ed.] 9781780841892, 9781780841915

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Gout Nicola Dalbeth, Fernando Perez-Ruiz & Naomi Schlesinger

Gout Editors Nicola Dalbeth University of Auckland, Auckland, New Zealand Fernando Perez-Ruiz Hospital Universitario Cruces, Biscay, Spain Naomi Schlesinger University of Medicine and Dentistry of New Jersey, ­Robert Wood Johnson Medical School, NJ, USA

Published by Future Medicine Ltd Future Medicine Ltd, Unitec House, 2 Albert Place, London N3 1QB, UK www.futuremedicine.com ISSN: 2047-332X ISBN: 978-1-78084-191-5 (print) ISBN: 978-1-78084-190-8 (epub) ISBN: 978-1-78084-189-2 (pdf) © 2013 Future Medicine Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder. British Library Cataloguing-in-Publication Data. A catalogue record for this book is available from the British Library. Although the author and publisher have made every effort to ensure accuracy of published drug doses and other medical information, they take no responsibility for errors, omissions, or for any outcomes related to the book contents and take no responsibility for the use of any products described within the book. No claims or endorsements are made for any marketed drug or putative therapeutic agent under clinical investigation. Any product mentioned in the book should be used in accordance with the prescribing information prepared by the manufacturers, and ultimate responsibility rests with the prescribing physician. Content Development Editor: Duc Hong Le Senior Manager, Production & Design: Karen Rowland Head of Production: Philip Chapman Junior Managing Production Editor: Harriet Penny Production Editor: Georgia Patey Assistant Production Editors: Samantha Whitham, Abigail Baxter & Kirsty Brown Editorial Assistant: Ben Kempson Graphics & Design Manager: Hannah Morton

Contents Gout Nicola Dalbeth, Fernando Perez-Ruiz & Naomi Schlesinger Epidemiology of gout Yong Gil Hwang & Kenneth G Saag Pathological basis of hyperuricemia and gout Nicola Dalbeth Pathophysiology and immunology of acute and chronic gout Ru Liu-Bryan An update on the genetic causes of hyperuricemia and gout Tony R Merriman & Cushla McKinney Clinical features of gout Naomi Schlesinger Synovial fluid analysis and crystal identification Eliseo Pascual & Mariano Andrés Imaging of gout Ralf G Thiele Outcome measures in gout Puja P Khanna & Dinesh Khanna The patient’s perspective of gout Romy Aranguiz & Leslie R Harrold

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Contents Continued

Asymptomatic hyperuricemia and its clinical implications for extra-articular disease Angelo L Gaffo Principles of gout management Fernando Perez-Ruiz & Ana M Herrero-Beites Urate-lowering therapy: xanthine oxidase inhibitors Lisa K Stamp Urate-lowering therapy: uricosurics Richard O Day, Kenneth M Williams & Garry G Graham Urate-lowering therapy: uricases Fernando Perez-Ruiz, Ane Altuna & Joana Atxotegi Therapies for acute flares and gout flare prophylaxis Naomi Schlesinger Gout comorbidities: prevalence and management Miguel Martillo, Elaine Karis, Daria B Crittenden & Michael H Pillinger Emerging therapeutics for acute and chronic gout Saima Chohan & Michael A Becker Index

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About the Editors Nicola Dalbeth Nicola Dalbeth is a Consultant Rheumatologist and Associate Professor of Medicine at the University of Auckland (New Zealand). She is a Principal Investigator in the Auckland Bone and Joint Research Group. She leads a clinical and laboratory research program in gout, focusing on the mechanisms of inflammation and joint damage in chronic gout.

Fernando Perez-Ruiz Fernando Perez-Ruiz is a Senior Clinician at the Hospital Universitario Cruces (Biscay, Spain). His clinical research over the last 20 years has been focused on treatment, outcome measures, audits and recommendations for gout.

Naomi Schlesinger Naomi Schlesinger is Chief of the Division of Rheumatology and Rheumatology Fellowship Program Director at the University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School (USA), where she is also Professor of Medicine in the Department of Medicine. She is a noted authority in the field of gout, having published papers regarding the diagnosis, treatment and better understanding of the pathogenesis of gout. She is the author of over 190 scientific articles, abstracts, book chapters and reviews. She is the past President of the New Jersey Rheumatology Association. In addition she is Co-Chair of the American College of Rheumatology Crystal Study Group, as well as the Co-Chair of the American College of Rheumatology Abstract Review: Metabolic and Crystal Arthropathies. She has a special interest in evidencebased medicine and serves as a cofacilitator of the Acute Gout Review Group for Cochrane International Collaboration as well as a member of the Outcome Measures in Rheumatology Gout Special Interest Organizing Group.

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Foreword Gout

Nicola Dalbeth, Fernando Perez-Ruiz & Naomi Schlesinger In the last decade, we have witnessed exciting advances in the understanding and treatment of gout. Gout is now the most common inflammatory arthritis, affecting almost 4% of the US population, with increasing rates in the last two decades [1]. The impact of poorly controlled gout on health-related quality of life, disability, work, participation and healthcare utilization has been highlighted, emphasizing the need for improved management of this condition. We have also seen new understanding of the pathophysiology of gout, particularly the genetics of hyperuricemia, environmental risk factors and the underlying immunological basis of this inflammatory disease. The complex relationship between hyperuricemia/gout and comorbid conditions has been explored. Imaging methods, particularly ultrasonography and dual energy computed tomography have allowed us to understand the mechanisms of disease in new ways. Large international collaborations have led to important progress in definitions, outcome measures for clinical trials and the genetic basis of gout [2–4]. The application of these scientific discoveries is leading to new therapeutic strategies to combat both acute gout flares and prevent the consequences of poorly controlled disease. The identification that activation of the NLRP3 inflammasome and subsequent release of mature IL-1b is central to the initiation phase of acute gout inflammation [5] has led to the identification of anti-IL-1 biologic therapy for treatment and prophylaxis doi:10.2217/EBO.12.529

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Dalbeth, Perez-Ruiz & Schlesinger of acute flares [6]. Two new urate-lowering agents have been approved by the US FDA in the last 5 years, febuxostat and pegloticase [7,8], and a number of other novel urate-lowering therapies are in Phase III clinical trials. In addition to these new agents, the optimal use of ‘veteran’ drugs such as colchicine and allopurinol has been clarified. Gout management guidelines have been published by the major rheumatology societies, including the European League Against Rheumatism, British Society for Rheumatology and American College of Rheumatology. Central to all of these guidelines is the importance of ‘treating to serum urate target’, a key strategy to address the underlying basis of disease and prevent the consequences of poorly controlled gout. This book highlights many of the advances in understanding mechanisms of disease, and provides a practical framework for gout management, outlining the therapeutic principles of gout management and providing detailed information about specific agents. Current controversies are explored, particularly issues around the relationship between serum urate and comorbid conditions, and management of ‘asymptomatic’ hyperuricemia. Despite the major advances described in this book, the quality of gout treatment is frequently poor. Understanding barriers to effective gout care and strategies to address these barriers is required to translate progress into therapeutic benefits for the patient. We hope that this book will provide a useful resource for researchers and clinicians, and ultimately lead to improved care of patients with gout. Financial & competing interests disclosure N Dalbeth has received consulting fees from Takeda, Ardea, Novartis, Fonterra and Metabolex; speaker’s fees from Novartis and Savient; and grant support from Fonterra. F Perez-Ruiz discloses consultancies/speakers bureau/educational materials/advisory board for Ardea, Menarini, Metabolex, Novartis and Savient. N Schlesinger reports having received a grant, travel expenses and payment for advisory board membership from Novartis Pharma, payment for advisory board membership and educational presentations from Takeda and Savient, and payment for advisory board membership from Savient, URL Pharma and Enzyme Rx. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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Gout References 1

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Zhu Y, Pandya BJ, Choi H. Prevalence of gout and hyperuricemia in the US general population: the National Health and Nutrition Examination Survey 2007–2008. Arthritis Rheum. 63(10), 3136–3141 (2011).

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Gaffo AL, Schumacher HR, Saag KG et al. Developing a provisional definition of flare in patients with established gout. Arthritis Rheum. 64(5), 1508–1517 (2012).

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Schumacher HR, Taylor W, Edwards L et al. Outcome domains for studies of acute and chronic gout. J. Rheumatol. 36(10), 2342–2345 (2009).

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ranging study. Arthritis Rheum. 62(10), 3064–3076 (2010).

Kolz M, Johnson T, Sanna S et al. Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations. PLoS Genet. 5(6), e1000504 (2009).

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Martinon F, Pétrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440(7081), 237–241 (2006).

Becker MA, Schumacher HR Jr, Wortmann RL et al. Febuxostat compared with allopurinol in patients with hyperuricemia and gout. N. Engl. J. Med. 353(23), 2450–2461 (2005).

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Sundy JS, Baraf HS, Yood RA et al. Efficacy and tolerability of pegloticase for the treatment of chronic gout in patients refractory to conventional treatment: two randomized controlled trials. JAMA 306(7), 711–720 (2011).

So A, De Meulemeester M, Pikhlak A et al. Canakinumab for the treatment of acute flares in difficult-to-treat gouty arthritis: results of a multicenter, Phase II, dose-

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About the Authors Yong Gil Hwang Yong Gil Hwang recently completed a clinical immunology and rheumatology fellowship at the University of Alabama at Bimingham (USA). He graduated from the Univeristy of Ulsan, College of Medicine (Seoul, South Korea) and completed a residency in internal medicine at the University of Texas Medical Branch at Galveston (USA), and a geriatric fellowship at the Cleveland Clinic (OH, USA).

Kenneth G Saag Kenneth G Saag is Lowe Professor of Medicine in the Division of Clinical Immunology and Rheumatology, at the University of Alabama at Birmingham. He is Director of the University of Alabama at Birmingham Center for Education and Research on Therapeutics, the Center for Outcomes and Effectiveness Research and Education, and the Center of Research Translation in Gout and Hyperuricemana. His research focuses on the epidemiology and outcomes of gout. He currently serves on the Board of Directors of the American Gout Society and is Chairman of the Quality of Care Committee for the American College of Rheumatology.

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Chapter

Epidemiology of gout

Incidence & prevalence: gout has been on the rise for decades

Yong Gil Hwang & Kenneth G Saag 8

Risk factors & comorbidities 

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Mortality

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Epidemiologic studies suggest that prevalence and incidence of gout have risen in recent decades. The rise in gout prevalence is primarily associated with an aging population, but it is also associated with a changing pattern of risk factors such as changes in diet, the obesity epidemic and increased rates of comorbidities associated with hyper­ uricemia and gout. Increased use of medications to treat such comorbidities may contribute to the rise in gout prevalence. With the increase in prevalence of gout, treatment imposes a significant economic burden, especially among the elderly population.

doi:10.2217/EBO.12.223

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Hwang & Saag Numerous risk factors for the development of gout have been established through recent epidemiologic studies. There are both nonmodifiable and modifiable risk factors for hyperuricemia and gout. Nonmodifiable Hyperuricemia: a serum urate level more than two risk factors include genetic factors, age and standard deviations from the mean in a gender- and sex. Several renal urate transporters have age-matched healthy population, in population-based terms; any level above 6.8 mg/dl is hyperuricemia in been identified, and polymorphisms in these physiological terms because serum is supersaturated genes are associated with an increased risk for monosodium urate at concentrations above [1]. of hyperuricemia and gout. Although gout is more common in men and is strongly agerelated, there is a narrower gender gap among elderly patients. Modifiable risk factors include dietary factors, the use of certain medications (e.g., diuretics, low-dose aspirin or cyclosporine) and comorbidities including metabolic syndrome and impaired renal function. Recent studies have provided information on dietary risk factors, including: higher intake of red meat, sugar-sweetened soft drinks, consumption of foods high in fructose and consumption of alcoholic beverages, especially beer and hard liquor. Comorbidities including renal insufficiency, hypertension and metabolic syndrome are common in gout patients; however, the causal relationship between gout and these comorbidities remains uncertain. Gout: an excruciatingly painful inflammatory arthritis, triggered by the crystallization of monosodium urate in soft tissues and joints, leading to an acute inflammatory response, a chronic destructive arthropathy and/or formation of tophi.

The association between gout and all-cause/cardiovascular mortality has been debated. It is unclear whether these associations are independent of traditional cardiovascular risk factors. Recent evidence suggests that gout (and/or hyperuricemia) may be an independent risk factor for all-cause mortality and cardiovascular mortality, additional to the risk conferred by its association with traditional cardiovascular risk factors.

Incidence & prevalence: gout has been on the rise for decades Pitfalls of using epidemiologic data in gout The gold standard for gout diagnosis is microscopic identification of monosodium urate crystals and clinical diagnosis has been shown to have poor sensitivity and specificity compared with this method [1,2]. In addition, given the episodic nature of gout, assessing the incidence and prevalence of gout at the population level is challenging. The diagnosis of gout, in the vast majority of Nonmodifiable and modifiable risk factors in epidemiologic studies, is based upon clinical conjunction with coexisting comorbidities assessment, patient self-report, general (e.g., renal insufficiency, hypertension and metabolic syndrome) contribute to the development of practice diagnosis, medical record/database hyperuricemia and gout. review or fulfillment of the 1977 American Gout is associated with increased all-cause mortality Rheumatism Association preliminary criteria and cardiovascular disease mortality.

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Epidemiology of gout for the acute arthritis of primary gout [3]. Although gout estimates vary due to variations in methodology between studies and the population being described, an increase in prevalence in recent decades has been noted worldwide. Prevalence of gout The prevalence of gout in the USA more than doubled between the 1960s and the 1990s [4]. According to the latest nationally representative sample of US men and women (National Health and Nutrition Examination Survey [NHANES]; 2007–2008), the prevalence of self-reported physiciandiagnosed gout among adults from the USA from 2007 to 2008 was 3.9% (8.3 million individuals). The prevalence among men was 5.9% (6.1 million), and the prevalence among women was 2.0% (2.2 million). The prevalence of gout increased with age, with the lowest prevalence (0.4% [0.2 million]) in individuals aged 20–29  years and the highest prevalence (12.6% [1.2 million]) among those aged 80 years or older. These estimates were significantly higher than the prevalence estimate in NHANES-III (1988–1994; 2.7% [95% CI: 2.3–3.0]), with a difference of 1.2% (95% CI: 0.6–1.9), suggesting that gout has been on the rise for the past two decades [5]. Epidemiological surveys undertaken in general practices in the UK also suggest that gout is becoming more prevalent [6]. A recent retrospective study of patients with gout, identified through the records of 2.5 million patients in UK general practices and 2.4  million patients attending general practitioners or internists in Germany, showed the same 1.4% prevalence of gout in both countries from 2000 to 2005, consistent with previous UK General Practice Research Database (GPRD) for the years 1990–1999 [7,8]. Incidence of gout In the Framingham Heart Study, which followed 5209 people over a 52-year period (1950–2002) prospectively, the incidence of gout was 1.4 per 1000 person-years in women and 4.0 per 1000 person-years in men [9]. The Rochester Epidemiology Project has reported that the incidence of gout has doubled over time. Comparing two successive surveys from the USA (MN, USA) during the time intervals of 1977–1978 and 1995–1996, respectively, the age- and sex-adjusted annual incidence rate rose from 20.2/100,000 to 45.9/100,000 individuals. However, the incidence of secondary, diuretic-related gout did not increase over time [10]. The yearly incidence rates for the UK, derived from the GPRD for 1990–1999, showed a modest increase in the early

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Prevalence and incidence of gout has increased worldwide in recent decades, largely due to an aging population and changes in gout risk factors.

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Hwang & Saag 1990s, but the overall gout incidence remained relatively stable, ranging from a low of 11.9 cases per 10,000 patient years in 1991 to a high of 18.0 cases per 10,000 patient-years in 1994 [7]. A retrospective study from the Royal College of General Practitioners Weekly Returns Service surveillance data in England and Wales between 1994 and 2007 showed that the mean annual incidence was 18.6 per 10,000 individuals, stable over the period [11]. In a later study using The Health Improvement Network UK primary care database between 2000 and 2007, the incidence of gout per 1000 person-years was estimated as 2.68 (4.42 in men and 1.32 in women) and increased with age [12]. Healthcare costs related to gout With increases in the prevalence of gout, treatment of gout impose a significant economic burden, especially among the elderly population [13]. According to a recent study using the Integrated Healthcare Information Services claims database (1999–2005), the average total healthcare cost per gouty flare was US$3096. The average gout-related cost per gouty flare was US$520 during the 30-day period following the onset of a flare [14].

Risk factors & comorbidities The rise in gout prevalence is primarily associated with an aging population, but it may also be associated with changes in gout risk factors, such as changes in diet, obesity epidemic and the increasing prevalence of comorbidities, such as metabolic syndrome, hypertension and advanced chronic kidney disease. Increased use of diuretics as a first-line therapy for hypertension and the wide use of low-dose aspirin for the prevention of cardiovascular disease (CVD) may contribute to the rise in gout prevalence. Patients with gout typically harbor multiple comorbidities, and these comorbidities have a strong association with gout. The causal relationship between gout and these comorbidities remains uncertain. Nonmodifiable risk factors Genetic factors

Primary gout in men often shows a strong familial predisposition, and twin studies have shown high heritability for both uric acid renal clearance (60%) and uric acid:creatinine ratio (87%) [15]. Several renal urate transporters have been identified, and polymorphisms in these genes are associated with an increased risk of hyperuricemia and gout. These genes include SLC2A9 and ABCG2 (discussed in detail in Chapter 4). Gout prevalence varies Uric acid: the end product of purine nucleotide substantially by geographic region, metabolism, which exists largely in the form of

urate at physiologic pH.

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Epidemiology of gout suggesting both genetic and environmental factors predispose individuals to developing gout [16,17]. Age

The incidence and prevalence of gout increase substantially with age in both men and women (Figure 1.1) [7]. This is probably due to multiple factors, including higher prevalence of age-associated diseases, such as metabolic syndrome, hypertension, chronic kidney disease and wide exposure to treatments that raise serum urate levels (e.g., thiazide diuretics) [18]. Sex

Gout has historically been considered a male disease. However, the incidence of gout has doubled among women over the past 20 years and gout in the elderly has a narrower gender gap [7]. Increasing age, obesity, alcohol consumption, hypertension and diuretic use were associated with the risk of incident gout among women and the magnitudes of associations Figure 1.1. Gout prevalence (1999) among enrollees in the UK General Practice Research Database, demonstrating that it is predominantly a disease of older men. 100

Prevalence (per 1000 patients)

90 80

Men Women Total

70 60 50 40 30 20 10 0

85

Age group (years) Gout becomes more common in women after the menopause. Bars indicate 95% CI calculated using normal approximation. Reproduced with permission from the BMJ Publishing Group Ltd [7].

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Hwang & Saag with these factors did not differ significantly from those among men, except for a stronger age effect among women than men, probably reflecting the loss of the uricosuric effect of estrogen following the menopause [9]. Modifiable risk factors Hyperuricemia

Hyperuricemia, a state of elevated high serum urate level, is considered the most important risk factor for the development of gout. Large proportions of people, men in particular, have urate levels above the solubility threshold of approximately 6.8 mg/dl and, depending on the definition, a prevalence of up to 15–20% has been reported in population-based studies [7]. A cohort of 2046 male veterans in the Normative Aging Study was followed over a period of 15 years. For those with baseline serum urate levels of 9 mg/dl or more, the annual incidence rate of gouty arthritis was 4.9%, compared with 0.5% for urate levels of 7.0–8.9 mg/dl and 0.1% for urate levels below 7.0 mg/dl. With urate levels of 9 mg/dl or higher, cumulative incidence of gouty arthritis reached 22% after 5 years [19]. In the Framingham Heart Study, the risk of developing gout increased similarly with increasing serum urate levels in both men and women, but the magnitude of this association was lower among women than men (Figure 1.2) [9]. In addition, in patients with established gout, reduction of serum urate concentrations to 6  mg/dl or lower will eventually result in a reduced frequency or prevention of future gouty attacks, and patients who do not achieve target serum urate are at increased flare risk [20,21]. Diet

An association between gout and purine-rich foods such as meats, seafood, purine-rich vegetables and high protein intake has been recognized for centuries. However, it is only recently that large, well-designed, prospective epidemiological studies have been undertaken (Table 1.1) [22,23]. In the Health Professionals Follow-Up Study (HPFS), the relationship between these purported dietary risk factors and the incidence of gout during a 12-year period in 47,150 male health professionals participants (730 incident gout cases) were examined prospectively, documenting 757 incident cases of gout [22,23]. Diet was assessed using the Willett Food Frequency Questionnaire, an instrument that is the ‘gold standard’ in nutritional epidemiology. After adjustment for age, BMI, diuretic use, hypertension, renal failure, alcohol intake and other dietary factors, increased meat and seafood intake were associated with increased risk for gout; however, no increase in risk for gout was associated with the intake of purine-rich vegetables or total protein intake. There was a 50% reduction

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Epidemiology of gout Figure 1.2. Relationship between serum uric acid levels and annual incidence of gout in the Framingham Heart Study cohort (women: 56% [mean age: 47 years]; men: 44% [mean age: 46 years]). 4

Annual incidence of gout (%)

Women Men 3

2 p = 0.0002

1

0

8.0

Serum uric acid (mg/dl) Over a 28-year median follow-up period, the incidence of gout was 1.4 per 1000 person-years in women and 4.0 per 1000 person-years in men. The incidence of gout increased with increasing levels of serum uric acid among women, but the rate of increase was lower than men. Reproduced with permission from John Wiley & Sons Ltd [9].

in gout incidence in persons in the highest quintile of low-fat dairy products compared with those in the lowest quintile but not for high-fat dairy products. In a subsequent ana­lysis of the HPFS, the same investigators showed consumption of six or more cups of coffee per day is protective against the development of gout compared with no coffee consumption. Tea consumption and total caffeine intake were not associated with development of gout [24]. Subsequently, the authors demonstrated consumption of two or more sugar-sweetened soft drinks per day was associated with increased risk for the development of gout compared with less than one per month. In addition, increased total fructose intake was a risk factor for incident gout [25]. Recently, the authors reported that the incidence of gout decreased with increasing total vitamin C intake, possibly related to the uricosuric effect of vitamin C. The multivariate relative risk (RR) per 500  mg increase in total daily vitamin C intake was 0.83 (95% CI: 0.77–0.90). Compared with men who did not use supplemental

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14 Total caffeine† Decaffeinated coffee‡ Diet soft drinks§ Wine

1.81 (1.31–2.50)

2.53 (1.73–3.70)

2.51 (1.77–3.55)

1.60 (1.19–2.16)

Total fructose†

Total alcohol (≥50 g/day versus none)

Beer§

†

Highest versus lowest quintile. ‡ ≥4 cups/day versus none. § ≥2 drinks/day versus none. RR: Relative risk. Data from [22–26].

Spirits

§

§

Tea‡

1.85 (1.08–3.16)

Sugar-sweetened soft drinks§

†

1.05 (0.64–1.72) 　

　

　

0.73 (0.46–1.17)

1.12 (0.82–1.52)

Total vitamin C (≥1500 mg vs 3 g/day) aspirin is uricosuric, whereas at low dosages (1–2 g/day) it causes uric acid retention. Although the increase in serum urate level is very small at low dosages, the effect could be significant in the elderly population [32]. Hyperuricemia and gout are common complications of organ transplantation. Among renal transplant patients, 13% experienced newonset gout and as many as 50% became hyperuricemic [33]. Hyperuricemia in the setting of transplantation is often secondary to a combination of calcineurin inhibitor use, diuretics and renal insufficiency, including delayed allograft function. Cyclosporine, a calcineurin inhibitor, is thought to increase urate levels by reducing tubular uric acid secretion and lowering glomerular filtration rates owing to its ability to increase renal arterial vasoconstriction [34]. The hyperuricemia induced by cyclosporine is not restricted to renal transplant recipients, it is also frequent in heart or heart–lung transplant patients. Tacrolimus has the potential to increase

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Hwang & Saag urate through its effect on glomerular filtration rate, although there was a lower risk of new onset gout compared with cyclosporine in a retrospective study [35]. Comorbidities Renal disease

The association between hyperuricemia, gout and renal disease has long been recognized, each being a risk factor for the other. Clearly, hyperuricemia is a marker of renal dysfunction and chronic renal disease is an important risk factor for gout. It was associated with gout in both the HPFS (RR: 3.61; 95% CI: 1.60–8.14, adjusted for multiple confounders including diuretic use)  [18] and the UK GPRD (age- and sex-adjusted odds ratio [OR]: 4.95; 95% CI: 4.28–5.72) [7]. On the other hand, recent data provide evidence that high serum urate may lead to declines in glomerular filtration rates and can mediate hypertension. In a recent post hoc ana­lysis of the FOCUS study, maintenance or improvement in estimated glomerular filtration rate (eGFR) was inversely correlated with the quantitative reduction in serum urate from baseline in 116 hyperuricemic gout subjects who received daily doses of febuxostat for up to 5 years. For every 1 mg/dl decrease in serum urate, the model projected an expected improvement in eGFR of 1 ml/min from the untreated value [36]. In a prospective, randomized trial of 113 chronic kidney disease patients (eGFR: 10,000  leukocytes/mm2 (and frequently much higher cell numbers). PMNs predominate, usually >80% of leukocytes. MSU crystals are virtually always present within synovial fluid at the time of the acute gout attack, and are visualized as negatively bifrefringent, needle-shaped crystals of Acute gouty attacks are characterized by an variable length (Chapter  6) [4] . These intense neutrophilic synovitis.

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Pathological basis of hyperuricemia & gout crystals can be visualized both within the PMNs and as free crystals, unassociated with cells.

Pathology of tophaceous gout Clinically apparent tophi develop in some, but not all, patients with gout. These lesions represent a chronic granulomatous response to MSU crystals [18]. Tophi may be present within the synovium (often associated with chronic gouty synovitis as shown in Figure 2.3), within soft-tissue joint structures, such as tendons and bursae, or in extra-articular tissues, particularly skin [16]. Microscopically, tophi appear as chronic granulomatous lesions comprising collections of mononucleated and multinucleated macrophages surrounding a core of MSU monohydrate crystals and encased by dense connective tissue (Figure 2.4) [18,19]. MSU crystals are arranged in small compact clusters (Figure 2.5) [16]. Various zones have been identified within the tophus: the central crystalline core, the cellular corona zone surrounding the central core, and the outer fibrovascular zone. These lesions are organized structures involving both innate and adaptive immune Figure 2.3. Chronic gouty synovitis.

100 µm Inflamed proliferative synovium from the ring finger proximal interphalangeal joint in a patient with chronic gout. Hematoxylin and eosin staining.

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Dalbeth cells [20]. A cellular model of the tophus is shown in Figure 2.6. Numerous CD68+ mononucleated and multinucleated cells are present within the corona zone (Figure 2.4). Mast cells are present throughout the corona and fibrovascular zones. By contrast, PMNs are rarely observed. Plasma cells are present in very high numbers within the corona zone. Although fewer B cells are observed, B-cell aggregates are not infrequently present in the fibrovascular zone. Cells within the corona zone produce proinflammatory soluble factors, including IL-1b, TNF-a, S100A8 and matrix metalloprotease (MMP)-3 [19,20]. TGF-b1 is also produced by cells within the tophus [20]. The co-expression of IL-1b and TGF-b1 suggests that both pro- and antiinflammatory factors present within the tophus contribute to a cycle of chronic inflammation, attempted resolution and tissue remodeling.

Joint damage in gout Structural joint damage is a feature of advanced gout. Bone erosion is most frequently observed, with cartilage damage a later feature. The appearance Figure 2.4. CD68 staining of the gouty tophus.

*

* 10 µm Positively staining multinucleated and mononucleated cells predominate within the corona zone of the tophus. Asterisks indicate the central core of monosodium urate crystals within the tophus. Reproduced with permission from [20].

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Pathological basis of hyperuricemia & gout Figure 2.5. Collections of monosodium urate crystals within a tophus.

400 µm Unstained slide of a tophus under polarizing light microscopy with a red compensator.

of bone erosion in gout differs from other forms of erosive arthritis, as it is well defined, lacks associated peri-articular osteopenia and is often associated with new bone formation. Imaging and patho­logical studies have strongly implicated the tophus in the development of bone erosion in gout. At the bone–tophus interface, there are numerous bone-resorbing osteoclasts, and reduced numbers of bone-forming osteoblasts (Figure 2.7) [21]. MSU crystals have profound inhibitory effects on osteoblast survival and function and may also indirectly promote osteoclastogenesis through alteration of the receptor activator of NF-kB ligand (RANKL)/osteoprotegerin axis in osteo­blasts [21,22]. RANKL expressed by T cells within the tophus may further promote osteoclast formation and contribute to bone erosion in gout [23]. In addition to the effects of tophi on bone, chronic synovitis may also contribute to the development of bone erosion in gout. The causes of cartilage loss and other structural changes in advanced gout are not as well understood. Analysis of articular cartilage changes induced by MSU crystals is often complicated by the presence of concomitant osteoarthritis [8] . MSU crystals induce catabolic pathways in chondrocytes [24], and also impact negatively on chondrocyte viability and

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Dalbeth Figure 2.6. Cellular model of the gouty tophus. Fibrovascular zone

Corona zone

T cell B cell

Plasma cell Macrophage

CD68+ MNC Mast cell

Neutrophil TRAP+ MNC

The central crystalline core is surrounded by a cellular corona zone, which is encased by a fibrovascular zone. MNC: Mononuclear cell. Reproduced with permission from [20].

matrix formation [25]. Microscopically, chondrocyte death can be observed adjacent to MSU crystals, and proteoglycan staining may be depleted. Changes in articular cartilage may be induced by direct contact of MSU crystals, or through interactions with inflamed synovial membrane in response to these crystals. Synovial fibroblasts produce a number of factors implicated in cartilage degradation on exposure to MSU crystals, including PGE2, collagenase and nitric oxide [26]. MSU crystals may directly deposit within tendons and ligaments, or induce tophus invasion into these structures [12,16]. Lysis and impaired function of tenocytes also occurs in response to MSU crystals [27].

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Pathological basis of hyperuricemia & gout Figure 2.7. Bone–tophus interface in a patient with tophaceous gout.

20 µm Immunohistochemistry for the cathepsin K, demonstrating positively stained multinucleated cells consistent with osteoclasts on bone surface. Reproduced with permission from [21].

Conclusion It is of great interest that most individuals with hyperuricemia do not develop gout [28], suggesting that hyperuricemia is necessary but not sufficient for the development of gout. There are several potential explanations for this observation; firstly, additional factors may be required for formation of MSU crystals, or alternatively, host responses to formed MSU crystals may influence the clinical presentation of gout. Similarly, it is unclear why some individuals form tophi, whereas others with persistent hypeuricemia and documented acute gout flares do not. Host responses to the MSU crystals or possibly urate burden may account for these variations in clinical presentation. These observations highlight a key point that even though MSU crystal deposition has been recognized for centuries as the cause of gout, there are many uncertainties about the additional factors that influence the clinical manifestations of disease.

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Dalbeth Financial & competing interests disclosure N Dalbeth has received consulting fees from Takeda, Ardea/AstraZeneca, Novartis, Fonterra and Metabolex; speaker fees from Menorini, Novartis and Savient; and grant support from Fonterra. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ Hyperuricemia is an essential checkpoint in the development of gout. ƒƒ Monosodium urate (MSU) monohydrate crystals form in some individuals when tissue urate levels increase above saturation concentrations. ƒƒ Renal underexcretion of uric acid is a particularly important factor in the control of serum urate concentrations. ƒƒ Serum urate concentrations are influenced by a balance of urate production and elimination. ƒƒ The clinical features of gout occur as a manifestation of the host response to MSU crystals. ƒƒ The gouty tophus represents a chronic foreign body granulomatous response to MSU crystals. ƒƒ Tophi are strongly implicated in the development of joint damage in gout.

References 1

2

3

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Loeb JN. The influence of temperature on the solubility of monosodium urate. Arthritis Rheum. 15(2), 189–192 (1972). McLean L, Becker MA. Etiology and pathogenesis of gout. In: Rheumatology. Hochberg MC, Silman AJ, Smolen JS, Weinblatt ME, Weisman MH (Eds). Mosby Elsevier, Philadelphia, PA. 1841–1857 (2011). Choi HK, Liu S, Curhan G. Intake of purine-rich foods, protein, and dairy products and relationship to serum levels of uric acid: the Third National Health and Nutrition Examination Survey. Arthritis Rheum. 52(1), 283–289 (2005). Kottgen A, Albrecht E, Teumer A et al. Genome-wide association analyses identify 18 new loci associated with serum urate concentrations.

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Evidence for a promoter of urate crystal formation in gouty synovial fluid. Ann. Rheum. Dis. 50(8), 558–561 (1991).

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Quinones Galvan A, Natali A, Baldi S et al. Effect of insulin on uric acid excretion in humans. Am. J. Physiol. 268(1 Pt 1), E1–E5 (1995).

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Muehleman C, Li J, Aigner T et al. Association between crystals and cartilage degeneration in the ankle. J. Rheumatol. 35(6), 1108–1117 (2008).

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K et al. A role of IgM antibodies in monosodium urate crystal formation and associated adjuvanticity. J. Immunol. 182(4), 1912–1918 (2009).

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LM, Solano C et al. Joint and tendon subclinical involvement suggestive of gouty arthritis in asymptomatic hyperuricemia: an ultrasound controlled study. Arthritis Res. Ther. 13(1), R4 (2011).

tumour necrosis factor-a and matrix metalloproteinases, and apoptosis of macrophages in gout tophi. Virchows Arch. 437(5), 534–539 (2000). 20 Dalbeth N, Pool B, Gamble GD

et al. Cellular characterization of the gouty tophus: a quantitative analysis. Arthritis Rheum. 62(5), 1549–1556 (2010).

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et al. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440(7081), 237–241 (2006).

21 Dalbeth N, Smith T, Nicolson B

et al. Enhanced osteoclastogenesis in patients with tophaceous gout: urate crystals promote osteoclast development through interactions with stromal cells. Arthritis Rheum. 58(6), 1854–1865 (2008).

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the synovial membrane in gout. Light and electron microscopic studies. Interpretation of crystals in electron micrographs. Arthritis Rheum. 18(Suppl. 6), S771–S782 (1975).

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Hasselbacher P et al. Induction of collagenase and prostaglandin synthesis in synovial fibroblasts treated with monosodium urate crystals. J. Pharm. Pharmacol. 33(6), 382–383 (1981).

23 Lee SJ, Nam KI, Jin HM et al.

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Bone destruction by receptor activator of nuclear factor kB ligand-expressing T cells in chronic gouty arthritis. Arthritis Res. Ther. 13(5), R164 (2011).

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PA. Development of the gout tophus. An hypothesis. Am. J. Clin. Pathol. 91(2), 190–195 (1989).

et al. Effects of monosodium urate crystals on chondrocyte viability and function; implications for development of cartilage damage in chronic gout. Arthritis Rheum. 63(10), S77 (2011).

27 Chhana A, Callon KE, Pool B

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et al. Monosodium urate crystals inhibit tenocyte viability and function: implications for periarticular involvement in chronic gout. Arthritis Rheum. 64(10), S60 (2012). DeLabry LO. Asymptomatic hyperuricemia. Risks and consequences in the Normative Aging Study. Am. J. Med. 82(3), 421–426 (1987).

29 Merriman TR, Dalbeth N. The

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About the Author Ru Liu-Bryan Ru Liu-Bryan is Associate Professor in the Division of Rheumatology, Allergy and Immunology, Department of Medicine, University of California, San Diego (USA). She received her PhD in biochemistry/molecular biology from Imperial College, London University (UK). Her research is mainly focused on understanding the molecular mechanisms of pathogenesis of gout and osteoarthritis.

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Chapter

3 Pathophysiology and immunology of acute and chronic gout Ru Liu-Bryan

Pathophysiology of acute gout

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Pathophysiology of chronic gout

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Conclusion

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Gout is a common, painful, inflammatory arthritis triggered by the crystallization of uric acid within joints. Monosodium urate crystals deposited in articular and periarticular tissues can cause acute joint inflammation. Recurrent acute intermittent flares can result in chronic gouty arthritis, which could lead to bone and cartilage destruction [1].

doi:10.2217/EBO.12.160

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Liu-Bryan Acute gout: characterized by severely intense pain caused by deposition of monosodium urate (MSU) crystals in the joint. Neutrophil influx into the synovium and joint fluids is the pathological hallmark of acute gout.

Pathophysiology of acute gout Acute gout is classically a recurrent, parox ysmal disease mediated by differentiated ‘professional’ phagocytes. Neutrophil influx into the synovium and joint fluid is the pathological hallmark of acute gout [1,2]. The interaction of monosodium urate (MSU) crystals with resident cells in the joint (principally synovial lining cells and mast cells) is believed to be the primary factor for triggering acute neutrophil ingress and paroxysms of gouty inflammation, since neutrophils are absent in the normal joint (Figure 3.1) [1,2]. Proinflammatory cytokines IL-1 and TNFa induced by MSU crystals, chemokines of CXCL8 and closely related ligands of CXCR2 synergistically drive neutrophil infiltration into joints, and cause subsequent endothelium adhesion molecule expression, such as E-selectin [2–4]. Monocytes are also recruited into the joint and differentiated into the inflammatory macrophage phenotype (M1) [5]. Neutrophils and M1 macrophages can take up MSU crystals in joints, leading to the release of soluble inflammatory mediators including calgranulins S100A8 and S100A9, which can further amplify acute gouty inflammation [6]. Critical role of innate immunity in acute gouty inflammation The interaction of MSU crystals with cells such as monocytes, macrophages and neutrophils can rapidly activate a variety of stress kinases such as PI3K, Src, Syk, Pyk2 and MAPKs [7], as well as transcriptional factors NF-kB and activator protein-1, which leads to the induction of expression of a broad array of inflammatory mediators, such as COX-2, TNF-a, IL-1 and IL-6, and the CXCR2-binding chemokines CXCL8 and CXCL1 (GROa) [2,8,9].

How do MSU crystals transduce cell signaling that eventually leads to induction of inflammatory mediators? Naked MSU crystals have a negatively charged and highly reactive surface that nonspecifically binds many plasma proteins [10] and also engages cell surface proteins, including the Fc receptor CD16, platelet [11] and leukocyte integrins (e.g., CD11b/CD18) [12]. Both the negativity of crystal surface charge and surface irregularity appear to be important Management of gout requires not only treatment of acute inflammation and pain, but determinants of the inflammatory potential also prevention of continued flares. Recent clinical of MSU crystals. MSU crystals are known to studies using three biologics targeting IL-1b including activate cells through phagocytosis that can anakinra (IL-1 receptor antagonist), rilonacept (IL-1 be greatly enhanced by opsonization by IgG trap) and canakinumab (monoclonal, humanized IL-1b or complement components, thus being neutralizing antibody) have shown therapeutic benefits in acute and chronic gout by reducing the classical immunology orientated [13] . pain and inflammation, as well as the risk of recurrent However, MSU crystals were shown to flares.

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NLRP3 inflammasome, caspase-1 activation

IL-1β maturation

Free MSU crystals

Complement/crystals C5b–C9 MAC formation

Tophus instability or remodeling

Free MSU crystals, either newly formed or from tophi remodeling, activate resident cells in the joint to induce proinflammatory mediators including cytokines and chemokines possibly via MyD88-dependent TLR2 and TLR4 and complement C5b–C9 MAC pathways. These inflammatory cytokines and chemokines cause infiltration of neutrophils (large amounts) and monocytes in the joint, which can be further activated by MSU crystals to release soluble inflammatory mediators, leading to amplification of inflammation. MSU crystals activate NLRP3 inflammasome to induce IL-1b maturation via caspase-1. Chymase from mast cells, and proteinase 3 and elastase from neutrophils, also contribute to IL-1b maturation. Once the differentiated macrophages became mature, they can take up the crystals and apoptotic neutrophils in the joint and release anti-inflammatory mediators, resulting in resolution of inflammation. MAC: Membrane attack complex; MSU: Monosodium urate; MyD88: Myosin differentiation factor 88; TLR: Toll-like receptor.

Resolution of inflammation Uptake of crystals and apoptotic neutrophils by mature noninflammatory M2, release of anti-inflammatory mediators

Neutrophils and monocytes Influx, adherence and activation

Endothelium activation (e.g., E-selctin)

Inflammatory mediators (e.g., IL-1β, TNF-α, IL-6 and CXCL8)

MyD88-dependent (CD14, TLR2 and TLR4)

Activation of resident cells in the joint Synovial lining cells, macrophages and mast cells

Amplication of inflammation Release of soluble inflammatory mediators, monocyte differentiation to inflammatory M1, further activation by soluble mediators

Neutrophil proteinase 3, elastase

Mast cell chymase

New MSU crystal formation

Figure 3.1. Cascades of the initiation, amplification and resolution of acute gouty arthritis.

Pathophysiology & immunology of acute & chronic gout

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Liu-Bryan The innate immune system acts as the first-line defense in our bodies. Cell signaling via the components of the innate immune system such as complement, Toll-like receptors and NLRP3 mediates MSU crystal-induced inflammatory responses, suggesting a critical role of innate immunity in acute gouty inflammation.

activate dendritic cells (DCs) via mechanismindependent opsonization and antibody binding by directly engaging the cell surface lipids, mainly cholesterol [14]. In particular, cholesterol depletion from the plasma membrane completely blocked the DCs response to MSU crystals [14].

There have been remarkable discoveries and evolution in defining the roles of innate immunity in mediating gouty inflammation in the last decade. The innate immune system acts as the first line of the host defense. It is known to comprise a range of receptors and soluble proteins including pattern recognition receptors and complement activation pathways that detect pathogens or products released by damaged tissue and dying cells. Complement in MSU crystal-induced inflammation

Both the classic and alternative complement pathways can be activated by MSU crystals in vitro, which lead to elaboration of C5a that act as leukocyte chemoattractant via C5 cleavage, catalyzed by the MSU crystal surface [15,16]. An experimental gouty arthritis study demonstrated that the local assembly of the C5b–C9 membrane attack complex plays a substantial role in acute inflammation in the C6-deficient rabbit [17]. Specifically, C6 deficiency was associated with less inflammatory responses to MSU crystals in vivo [17]. Toll-like receptors in MSU crystal-induced inflammation

Toll-like receptors (TLRs), the type I transmembrane receptors, are one family of the pattern recognition receptorss [18,19]. They are critical sensors of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [18,19]. MSU crystals were first reported to activate TLR2 signaling pathways that mediated nitric oxide generation in articular chondrocytes [20]. Both TLR2 and TLR4 were later demonstrated to be critical for the capacities of naked MSU crystals (under serum-free conditions) to induce macrophage phagocytosis of crystals and expression of proinflammatory cytokines including IL1b in  vitro and acute inflammatory Increased acute flares are seen in patients responses in vivo [21]. In addition, myeloid when urate-lowering therapy is initiated. This is because urate-lowering therapy induces rapid differentiation factor 88 (MyD88), the changes in serum urate level, which can cause tophus intracellular adaptor protein for TLR2 and remodeling by altering stability of tophi in the joint. TLR4, as well as IL-1 receptor (IL-1R), played In this context, the chemical or physical state of a major role in the capacity of macrophages existing MSU crystals is altered, and anti-inflammatory to phagocytose MSU crystals in vitro and crystal surface proteins may be disassociated. The remodeled MSU crystals then trigger inflammation acute MSU crystal-induced inflammation again via innate immune signaling.

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Pathophysiology & immunology of acute & chronic gout in  vivo in a murine air pouch model [21]. MyD88-dependent IL-1R, TLR2 and TLR4 pathways were also shown to be required for MSU crystal-induced inflammation in the mouse lung injury model [22]. However, the MyD88-dependent IL-1R pathway, and not the MyD88-independent TLR pathway, was reported to be necessary for MSU crystal-induced inflammation in the mouse peritonitis model [23].

NLRP3 inflammasome: a multiprotein complex composed of procaspase-1, apoptosisassociated speck-like protein containing a caspaseassociated recruitment domain and NLRP3, which can be activated by MSU crystals. Upon activation, procaspase-1 is recruited and activated. In turn, the activated caspase-1 activates the proform of IL-1b via proteolytic cleavage, facilitating secretion of the active IL-1b, a pivotal cytokine in acute gouty inflammation.

Expression of the shared TLR2 and TLR4 adaptor protein CD14, a glycosylphosphatidylinositol-anchored cell surface protein, was necessary to convert macrophage ingestion of MSU crystals from a noninflammatory event to an inflammatory event in vitro [24]. Coating of MSU crystals with CD14 partially reconstituted the proinflammatory potential of naked MSU crystals for CD14-knockout macrophages [24]; thus, CD14-knockout mice had a significantly decreased inflammatory response to MSU crystals in the air pouch synovitis model in vivo [24]. These findings suggest that MSU crystals act as DAMP, and that innate immune recognition of MSU crystals by CD14, TLR2 and TLR4, which are expressed in synovial lining cells, neutrophils, monocytes and macrophages, could be a determinant of the inflammatory potential of MSU crystals. In addition, soluble inflammatory mediators induced by MSU crystals such S100A8 and S100A9 can further amplify inflammation via the TLR4 signaling pathway [25]. MSU crystals have been observed to induce expression of the receptor triggering receptor expressed on myeloid cells-1 (TREM-1), a cell surfaceexpressed immunoglobulin superfamily protein, in phagocytes in vitro and in vivo [26,27]. Costimulation of resident peritoneal macrophages with MSU crystals and an anti-TREM-1 agonist antibody synergistically increased the production of both IL-1b and monocyte chemoattractant protein-1 compared with stimulation with crystals alone [26]. Engagement of TLRs such as TLR2 and TLR4 was shown to upregulate TREM-1 expression, which can lead to amplification of a variety of inflammatory responses [28,29]. NLRP3 inflammasome & IL-1b release in MSU crystal-induced inflammation

Only in recent years, IL-1b was confirmed as the pivotal cytokine in gouty inflammation in  vivo. Inflammatory responses to MSU crystals were significantly reduced in mice treated with IL-1 neutralizing antibodies, and in mice deficient in IL-1R or MyD88 [23]. Importantly, NLRP3 inflammasome was shown to mediate IL-1b processing and release in response to MSU crystals [30].

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Liu-Bryan The NLRP3 inflammasome, central to several autoinflammatory syndromes, is a multiprotein cytosolic complex composed of procaspase-1, the cytoplasmic protein ASC (apoptosis-associated speck-like protein containing a caspase-associated recruitment domain), and NLRP3 (also named NALP3, cryopyrin and CIAS1) [31]. It is assembled and activated in response to a large variety of soluble and particulate agonists. These include PAMPs (e.g., bacterial muramyl dipeptide, a degradation product of the bacterial cell-wall component peptidoglycan), microbial toxins, RNA of bacterial and viral origin, imidazoquinoline and cytosolic microbial and host DNA, and DAMPs (e.g., extracellular ATP released from dying cells and amyloid-b, MSU, calcium pyrophosphate dihydrate crystals, as well as cholesterol crystals, aluminium, asbestos and silica) [30–33]. Upon activation of the NLRP3 inflammasome, procaspase-1 is recruited and activated (via proteolytic cleavage). The activated caspase-1 in turn proteolytically cleaves and activates the proform of IL-1b, facilitating secretion of the active IL-1b [31–33]. Interestingly, many of these diverse PAMPs and DAMPs engage TLRs to induce synthesis of the proform of IL-1b through MyD88dependent signaling pathways, a priming step that is required for subsequent NLRP3 inflammasome activation. The exact mechanisms by which these diverse PAMPs and DAMPs trigger NLRP3 inflammasome activation are still unknown. However, potassium efflux, reactive oxygen species production and the release of cathespin B from the lysosme are common intermediates triggered by these activators [31–33]. Studies of mice with knockouts of caspase-1, ASC and NLRP3 itself as well as the NLRP3-mutant mice with a deletion of the entire leucine-rich repeat region (involved in sensing agonists and proper folding of the molecule), have revealed a central role of the NLRP3 inflammasome in experimental gouty inflammation [30,34]. MSU crystal-induced ATP release has also been suggested to amplify experimental gouty inflammation via P2X7 signaling [35]. NLRP3 was recently shown to interact with thioredoxininteracting protein (TXNIP). MSU crystals induced the dissociation of TXNIP from thioredoxin in a reactive oxygen species-sensitive manner and allowed binding to NLRP3 [36]. In addition, TXNIP deficiency impaired NLRP3 inflammasome and IL-1b release in macrophages in response to MSU crystals [36]. These data further support the notion that oxidative stress triggers NLRP3 inflammasome activation. It should be noted that NLRP3 inflammasome-independent processing of pro-IL-1b exists. Octacalcium crystals (a form of basic calcium phosphates) stimulate peritoneal inflammation in vivo via IL-1b-dependent and NLRP3independent mechanisms [37]. In addition, MSU crystals were recently shown to have synergistic effect with free fatty acids to induce IL-1b

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Pathophysiology & immunology of acute & chronic gout release dependent on ASC and caspase-1, but not NLRP3 in human peripheral blood mononuclear cells and murine macrophages [38]. Moreover, serine protease derived from mast cells (chymase) and neutrophils (proteinase 3 and neutrophil elastase) have also been demonstrated to contribute to processing of pro-IL-1b during acute inflammatory arthritis [39–41] in animal models. The discrepancies in results related to TLR2, TLR4 and NLRP3 in MSU crystal-induced animal models of gouty inflammation (mentioned above) are probably complex [21,23,38]. Differences in MSU crystal size and surface properties among various studies could be substantial. For instance, MSU crystals larger than the diameter of phagocytes (i.e., greater than 15 µm) are less inflammatory. In addition, there may be substantial differences in MSU crystal inflammation model systems (e.g., injection of crystals into joints, synovium-like subcutaneous air pouches, peritoneum and lung tissue). Different types of resident cells in these models (e.g., tissue macrophages or serosal surface fibroblasts) may have differential requirements for activation, and/or local factors (e.g., proteins) bound to crystals that could modify the cellular responses. Resolution of acute gouty inflammation Acute gouty inflammation is typically self-limited within 7–10  days. Neutralization and/or removal of MSU crystals, clearance of apoptotic cells and cellular debris, and a switch in the milieu of soluble mediators from proinflammatory to anti-inflammatory can occur during the process of resolution of acute inflammation [42]. The surface coat of MSU crystals undergoes dynamic alteration during acute gouty inflammation. MSU crystal-bound proteins could influence their inflammatory potential. MSU crystal-bound IgG, increases the capacity of the crystals to stimulate a variety of cells, and generally becomes less abundant on the crystal surface with time [42]. By contrast, MSU crystal-bound apolipoprotein  B increases as gouty inflammation starts to resolve [42]. Macrophages can avidly take up MSU crystals. This uptake has been shown to be dependent on the state of differentiation of the macrophages [42], with mature macrophages (M2 phenotype) taking up greater amounts of MSU crystals. Thus, mature macrophages presented later in acute gout may contribute to the resolution of gouty inflammation (see Figure 3.1). Cell surface receptors capable of engaging crystals may be responsible for effective clearance of MSU crystals. Efficient and rapid clearance of apoptotic neutrophils by macrophages and nonprofessional phagocytic cells are important for resolution of acute

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Liu-Bryan gouty inflammation. Defects in apoptotic cell clearance can lead to over exuberant and chronic inflammation, as well as predisposing to the development of autoimmunity in several animal models of inflammation [42]. Transglutaminase-2 has been demonstrated to play a role in antiinflammatory clearance of apoptotic cells by macrophages in response to MSU crystals, which is centrally involved in resolution of experimental gouty inflammation [43]. There is a switch in mediator profile from proinflammatory, present early in the course of the inflammation, to anti-inflammatory, which arise later over the course of gouty inflammation. The uptake of MSU crystals as well as apoptotic cells by macrophages can trigger production of the antiinflammatory mediators such as TGF-b, IL-10, prostaglandin  D2 and 15-deoxy-PGJ2 (ligand of PPARg), which are thought to play an important role in orchestrating the resolution of gouty inflammation [42]. For example, TGF-b suppresses endothelial E-selectin expression, which in turn reduces further neutrophil infiltration [42]. Prostaglandin D2 promotes neutrophil apoptosis and in conjunction with other lipid mediators can stimulate macrophage uptake of apoptotic cells [42]. Furthermore, TGF-b can promote fibroblast differentiation and fibrosis, which can contribute to wound repair and stabilization of MSU in tophi [42]. Increased production of IL-1ra and soluble TNF receptor I and II, as well as upregulation of intracellular cytokine-inducible SH2-containing protein and suppressors of cytokine signaling-3 protein expression are shown to associate with spontaneous resolution of gouty inflammation [44].

Pathophysiology of chronic gout Although acute gout is self-limited, if left untreated, the frequency and severity of flares may increase [45]. Over time, MSU crystals can be deposited into tophaceous, granuloma-like synovial microenvironments, and in macroaggregates near the articular cartilage surface, which can remain quiescent prior to and following flares of acute gout. Some microscopic tophi in the synovium have a macrophage-rich and fibroblast-rich ‘holding tank’ for MSU crystals lined by a ring of fibrinogen and other proteins. The predominant effect of serum protein binding to MSU crystals is that of physical suppression of the MSU crystal–cell interaction leading to decreased inflammation. There is an increase in acute flares when urate-lowering therapy (ULT) is initiated [46]. This may result from tophus remodeling, which alters the chemical or physical state of existing MSU crystals when ULT induces rapid changes in serum urate levels, leading to altered stability of tophi in the joint [46]. Decrease in the large size of MSU crystals via tophus remodeling,

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Pathophysiology & immunology of acute & chronic gout mechanical trauma that disrupts tophi or cartilage surface crystal macroaggregates and dissociation of anti-inflammatory crystal surface proteins are probably the triggers for acute flares. In this context, MSU crystals engage innate immune receptors (e.g., TLR2, TLR4 and NLRP3) to induce acute inflammatory responses as mentioned above. The activation of innate immunity can lead to the induction of the adaptive immune system through a process of antigen presentation. The nature of recurrent acute gout flares suggests that gouty inflammation is the result of classical innate immune ‘early induced’ response, which is quite distinct from adaptive immunity because there is no clear induction of ‘immunologic memory’ or lasting protective immunity. Interestingly, most recent studies showed that MSU crystals were capable of stimulating DCs, the antigen presenting cells, to promote release of cytokines IL-1a/b and IL-18 that drive Th17 differentiation [47]. Naive CD4+ T cells cocultured with MSU crystaltreated DCs produced a large amount of proinflammatory IL-17A [47]. This process was NLRP3 inflammasome dependent. Thus, deficiency in NLRP3, ASC or caspase-1 significantly impaired Th17 polarization [47]. A number of studies have demonstrated the critical pathogenic role of Th17 cells and its hallmark cytokine IL-17 in several autoimmune diseases including rheumatoid arthritis [47]. IL-17A can induce matrix metalloproteinases (MMPs), including MMP-3 and MMP-13, which play important roles in extracellular matrix destruction and tissue damage in rheumatoid arthritis [48]. MSU crystals have the capacity to induce cartilage degradation, demonstrated by induction of nitric oxide and MMP-3 expression by MSU crystals in articular chondrocytes in vitro [7,20]. Innate immune TLR2 signaling was shown to mediate MSU crystal-induced nitric oxide production in chondrocytes [20]. Increased nitric oxide generation inhibits chondrocyte proteoglycans synthesis, can impair chondrocyte viability and has the potential (through S-nitrosylation of certain MMPs) to enhance matrix catabolic activity of MMPs [7], leading to cartilage matrix degradation. Bone erosion is a common manifestation of chronic tophaceous gout. Under physiological conditions, the integrity of bone is maintained by the balanced activity of osteoblasts and osteoclasts, which are responsible for bone formation and resorption, respectively. MSU crystals can reduce osteoblast viability and differentiation, and enhance osteoclast development and activity in the vicinity of tophi. Patients with erosive tophaceous gout have fewer osteoblasts present on bone directly adjacent to tophus than bone unaffected by tophus [49], and have osteoclasts within tophi and at the interface between soft tissue and bone [50]. In addition, recent studies demonstrated that tophus tissue was also infiltrated with

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Liu-Bryan inflammatory T  cells, which express receptor activators of the NF-κB ligand, the master regulator of osteoclastogenesis, implicating their role in bone destruction [51]. Moreover, tophus-associated monocytes produce the proresorptive cytokines IL-1b, IL-6 and TNF-a, which could further promote osteoclast differentiation [51]. Interestingly, IL-17A also increases membrane expression of receptor activator of NF-κB ligand in osteoblasts, which in turn promotes osteoclastogenesis and subsequent bone destruction [48]. Further studies investigating whether IL-17 is a link between innate immunity and chronic gout is warranted.

Chronic gout: characterized by chronic destructive arthritis, often associated with the development of tophi and secondary degenerative changes such as bone erosion. Recurrent acute flares can progress to chronic gouty arthritis.

Conclusion Acute and chronic gout can have a great impact on patients’ quality of life due to severely intense pain, limitation of activity and disability. NSAIDs, colchicine and corticosteroids are the conventional treatment for acute and chronic gouty inflammation. The primary targets that these drugs act on are phagocyte-mediated inflammation and pain. Management of gout needs to not only treat acute inflammation and pain, but also prevent continued gouty flares. Recent advances in understanding the important role of NLRP3 inflammasome in MSU crystal-induced inflammation have led to the development of selective biologics targeting IL-1b production such as anakinra (IL-1R antagonist), rilonacept (IL-1 trap) and canakinumab (monoclonal, humanized IL-1b neutralizing antibody) [46]. Clinical studies of these three biologics have shown therapeutic benefit for patients with acute and chronic gout [46]. Importantly, these biologic therapies may provide treatment alternatives for patients in whom the conventional treatment is inappropriate owing to contradictions, unresponsiveness or intolerance to side effects. It has recently become clear that even when the patient is asymptomatic, chronic inflammation is often present in patients with chronic gouty arthritis. Selective blockade of IL-1b can reduce pain and inflammation, as well as the risk of recurrent flares [46]. Specifically, both rilonacept and canakinnumab have demonstrated significant flare prevention during ULT initiation in clinical trials [46], suggesting IL-1 antagonism as an effective prophylaxis approach. Given the recent novel finding that NLRP3 inflammasome-mediated innate immune responses to MSU crystals can potentially shape adaptive immunity (Th17 polarization), it would be interesting to determine whether this is sufficient to kick-start autoreactive T-cell responses, or whether it amplifies responses that are induced by

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Pathophysiology & immunology of acute & chronic gout MSU crystals. Whether IL-17 plays a role in pathogenesis of gout should be investigated. This would further advance our knowledge on pathophysiology of gout and may open new avenues for the development of novel therapeutic interventions. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ Innate immunity plays a critical role in acute gout. ƒƒ IL-1b is a pivotal cytokine in acute gouty inflammation. ƒƒ Recurrent acute flares can progress to chronic gout. ƒƒ NSAIDs, colchicine and corticosteroids are the conventional treatment for managing inflammation and pain in acute and chronic gout. ƒƒ Three IL-1b biologics including anakinra (IL-1 receptor antagonist), rilonacept (IL-1 trap), and canakinumab (monoclonal, humanized IL-1b neutralizing antibody) can reduce pain and inflammation, and the risk of recurrent flares; therefore have therapeutic potential as new treatments for acute and chronic gout. ƒƒ Adaptive immunity, particularly the role of IL-17, in pathogenesis of gout needs to be further investigated.

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About the Authors Tony R Merriman Tony R Merriman is Associate Professor at the University of Otago (Dunedin, New Zealand). His research focus is the genetic and environmental causes of gout in the major ethnic groups of New Zealand (European, Māori and Pacific Island).

Cushla McKinney Cushla McKinney is a Research Fellow at the University of Otago. Her research focus is the genetic causes of gout in the presence of hyperuricemia.

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Chapter

4 An update on the genetic causes of hyperuricemia and gout

Genetic variants in uric acid transporters57 Association of serum urate genes with neurological disease62 Genetics of acute gout in the presence of hyperuricemia 62 Future perspective

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Tony R Merriman & Cushla McKinney Acute gout revolves around two key checkpoints – renal clearance of uric acid and an innate immune system response to monosodium urate crystals. Genome-wide association studies have identified two genes that encode novel uric acid transporters, SLC2A9 and ABCG2, which together explain a significant proportion of variation in serum urate levels. Other loci of weaker effect include uric acid transporters URAT1, OAT4 and NPT1, in addition to GCKR, which implicates the metabolism of simple sugars in serum urate levels. By comparison, progress in understanding the genetic causes of acute gout in the presence of hyperuricemia has been very poor. A primary reason for this is the lack of large, well-phenotyped gout clinical sample sets that are suitable for genetic studies.

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Merriman & McKinney Two biological processes are central to gout – renal control of uric acid clearance and the innate immune system response to monosodium urate (MSU) crystals [1]. Twin studies have demonstrated that serum urate levels are heritable, with 60% of variation in renal clearance of uric acid explained by inherited genetic variants [2]. Given the fact that only a moderate proportion of people with hyperuricemia develop gout (in extreme hyperuricemia the 5-year cumulative incidence of gout is 22% [3]), other factors are clearly involved in acute gout. However, there are few data on heritability in gout per  se, with the only reported twin study reporting no detectable heritability (a caveat to this study was the very high gout prevalence of 11.6% in the 514 male twin pairs studied) [4]. In this chapter, we will review the recent genetic progress clarifying the molecular pathways controlling serum urate levels and hence risk of gout, with a focus on common non-Mendelian gout. Mendelian (familial) gout has previously been thoroughly reviewed [5]. In the absence of robust data on genetic factors controlling acute gout in the presence of hyperuricemia, we will review possible candidate genes.

Innate immune system: as opposed to the adaptive immune system, the innate immune system is the nonspecific arm of the immune system that defends hosts from infection.

Genome-wide association studies (GWASs) have allowed new insights into a range of complex phenotypes, caused by many genetic and environmental factors working together, each having a relatively small effect and not necessarily required for disease [6]. A GWAS employs technology that simultaneously Genome-wide association study (GWAS): a determines the genotypes of hundreds of very large sample set of cases and controls thousands of single nucleotide (>2000 each) is genotyped for approximately 1 million polymorphisms (SNPs) spread throughout genetic variants (including SNPs and structural the genome that assess the majority of changes) spread throughout the genome that capture common genetic variation. Aside from technical known common variation [7]. Very large challenges, the largest challenge is the multiple case–control sample sets are used to testing inherent in this approach, meaning that very account for the low effect size of the typical large sample sets are required to detect, and replicate, complex disease genetic risk factor (odds associations. GWASs are typically carried out by ratio [OR]: 2) in sample sets ascertained using American College of Rheumatology clinical criteria [17]. NZ Māori and, to a lesser extent, Pacific Island people living in NZ, are admixed populations, predominantly with the Caucasian population. A study of tightly linked groups of markers (haplotypes) suggests that the association at this SLC2A9 locus is driven by the inheritance of dominant protective genetic variants, probably derived from European–Caucasian ancestry [17]. It can be hypothesized that the protective haplotype reduces expression of the deleted isoform of SLC2A9 on the apical membrane of the renal tubule, thus reducing reuptake of filtered uric acid. There is no evidence for association of the intronic variants with gout in Japanese and Chinese studies [18,19], with the variant that confers risk in the other populations being very common (>97%) and nearly invariant (monomorphic). This means that variation in the intronic genetic variants is not playing a role in differentiating risk within these populations, although it can be assumed that the intronic SLC2A9 risk variant plays a role in gout. A second variant (Arg265His), genetically independent of the intronic variants, has been studied in non-Caucasian groups [18–20] . This nonsynonymous variant (Arg265His) has not been reported to influence SLC2A9 function and is not reported as associated with serum urate levels in Caucasians. Heterogeneity in association with gout is also evident at this variant. In a pattern inverse to that seen at the intronic variants, a significant, albeit weaker, genetic effect is observed in Han Chinese and Japanese (OR: ~1.5) [18,19], but not in NZ Caucasian, Māori or Pacific Island, sample sets [20]. Notably, there is large variation in frequency of the risk allele, from 32% in Asian populations to 81% in Caucasian populations, with the significant associations seen when the risk allele is relatively low. It has not yet been proven whether Arg263His is the causative variant in Han Chinese and Japanese – it could itself be in linkage disequilibrium (correlated) with the causative variant in the vicinity.

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Genetic causes of hyperuricemia & gout Other genes of weaker effect on serum urate ABCG2 levels and gout risk include genes encoding A GWAS also identified association of the renal uric acid transporters URAT1, OAT4 and NPT1, nonsynonymous Gln141Lys variant of the in addition to GCKR, which implicates the metabolism ABCG2  gene with serum urate levels in of simple sugars in serum urate levels. Caucasian patients [21]. Like SLC2A9, ABCG2 was a previously undiscovered urate transporter [22]. Unlike the situation with SLC2A9 where the etiological intronic variant has not been identified, the Q141K variant of ABGC2 is almost certainly the etiological variant, with the lysine allele associated with increased serum urate levels [22]. ABCG2 is highly expressed in intestinal epithelial cells and also expressed in the apical membrane of the kidney proximal tubule. ABCG2 is an ATPdependent uric acid secretory molecule with the lysine risk allele encoding a molecule with approximately 50% reduced ability to transport uric acid [22]. The Gln141Lys variant explains approximately 0.5% of the variation in serum urate levels – an effect size, while smaller than that of the intronic variants of SLC2A9, is large in the context of complex phenotypes.

The ABCG2 141Lys allele increases risk of gout in Caucasian, Chinese, Japanese and NZ Pacific sample sets (OR: ~2 [10,23–25]) but, for unclear reasons, not in NZ Māori, despite an allele frequency similar to Caucasian cases [23]. A rarer variant (Gln126Stop), which results in a truncated and inactive protein, is also a risk factor for gout in the Japanese population [24], with the role of this variant in gout risk in other populations currently not known. In the gut epithelium, ABCG2 secretes uric acid [26]. However, the direction of transport in the renal tubule is not clear. Ichida et al. classified people with hyperuricemia into four groups according to ABCG2 genotype, from full function (possessing the urate-lowering glutamine allele at each of Gln126Stop and Gln141Lys) to ≤25% of function (positivity for both the 126Stop and 141Lys alleles) [26]. Unexpectedly, individuals with a full function genotype were less able to excrete uric acid. Other urate transporters Weaker effects on serum urate levels have been identified by a GWAS – sodium phosphate transporter-1 (NPT1/SLC17A1), the urate transporter 1 (URAT1/SLC22A12) and organic anion transporter 4 (OAT4/SLC22A11) – and the PDZK1 gene, which encodes a molecule known to anchor renal transport molecules to the tubule cytoskeleton [10,27]. Of these, only SLC17A1 has been unequivocally associated with gout, with the same variant conferring a similar effect (OR: ~1.5) in Caucasian, Japanese and NZ Māori and Pacific Island people [8,28]. Despite a clear effect of variants within PDZK1 on serum urate levels, the same variants had no effect whatsoever on the risk of gout in a Caucasian

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Merriman & McKinney meta-analysis of cases nested within population-based cohorts (OR: 1.00) [10], nor was there association with gout in a German–Caucasian sample set [29]. Why this is the case is unclear; however, it is notable that the same allele of the rs12129861 SNP in PDZK1 that correlates with increased serum urate also correlates with decreased blood pressure [30]. Given that the gout cases studied by Yang et al. [10] and Stark et al. [29] included cases likely to be secondary to diuretic medication (for hypertension), it is possible that any increased risk of primary gout mediated by the allele of rs12129861 via increased serum urate levels was negated by inclusion of cases with gout secondary to hypertension treatment in which the other (serum urate-lowering) allele of rs12129861 was over-represented. The genes encoding SLC22A11 and SLC22A12 are located together on chromosome 11. Variants in each of the genes have previously been associated with serum urate concentration in Caucasians [10,31]. Given the lack of genetic correlation between these variants, these associations represent independent genetic effects. Their association with gout is currently equivocal, probably owing to a combination of inadequately powered studies and the use of heterogenous sample sets consisting of primary and secondary (to diuretic use) cases. SLC22A12 has previously been tested for association with gout in Caucasian patients on only one occasion, with Stark et al. reporting no evidence for association with gout [29], although up to 35% of the gout cases studied may have developed gout secondary to diuretic medication, which would be expected to over-ride any genetic effect on gout risk mediated by SLC22A12. The one report of association of SLC22A12 with gout in a Chinese sample set [32] is balanced by a report of no association [33]. At SLC22A11, Yang et al. reported association of weak effect with gout in a Caucasian meta-analysis of cases nested within population-based cohorts (OR: 1.26) [10]. However, Stark et al. reported no evidence for association of SLC22A11 with gout in a European–Caucasian sample set [29]. Glucokinase regulatory protein Other loci associated with serum urate levels by GWAS are SLC16A9, glucokinase regulatory protein (GCKR), INHBC and RREB1 [10,31]. Collectively, the ten loci discovered explain approximately 6% of the variation in serum urate levels. Of the SLC16A9, GCKR, INHBC and RREB1 loci, only GCKR and INHBC (encoding a member of the TGF-b superfamily) have been associated with gout [10,34]. The genetic variant maximally associated with serum urate levels at the INHBC locus maps halfway between the INHBC and RSHDM2 genes [10] – genetic fine-mapping is needed in order to determine which of these genes regulates serum urate levels. SLC16A9 encodes

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Genetic causes of hyperuricemia & gout monocarboxylic acid transporter 9 (MCT9). Kolz et al. postulate that, as other sodium monocarboxylate transporters have been found to influence urate levels in mouse knockout models, SLC16A9 may encode a sodiumdependent transporter in the kidney [31]. RREB1 encodes a transcription factor that regulates the androgen receptor [35]. The association with GCKR provides some clues to the etiological links between gout and other associated metabolic conditions such as diabetes and dyslipidemia [36]. Genetic variation in GCKR has also been associated with triglyceride and fasting glucose concentrations and risk of Type 2 diabetes [37–39]. Interestingly, the association of GCKR with serum urate levels reported by van der Harst et al. is attenuated by triglyceride levels, with the GCKR SNP (rs780094) also associated with triglyceride levels (the same allele was associated with increased serum urate and triglyceride) [30]. The most plausible explanation for this observation is that GCKR affects both serum urate and triglyceride levels by a common unconfirmed mediator, which could be glucose-6-phosphate [30]. GCKR controls the intracellular location and activity of glucokinase, and hence the hepatic production of glucose-6-phosphate, a precursor for de novo purine (uric acid) synthesis and the catabolic products of which are used for triglyceride synthesis via glycolysis, and pyruvate and acetyl CoA. GWAS in gout There have been two large GWASs performed with gout as the outcome measure. The first was carried out on approximately 1000 cases nested within the cohort meta-analyzed by Yang et al. for loci controlling serum urate [10]. The only genome-wide significant associations found with gout were SLC2A9 and ABCG2. A similar sized study in an Icelandic sample set detected genome-wide significant associations with gout at ABCG2 and ALDH16A1 (encoding an aldehyde dehydrogenase involved in the metabolism of alcohol) [40]. The ALDH16A1 association was driven by the minor allele of a rare genetic variant that protected from gout, which was present in 2% of the general population in Iceland and even rarer in other European–Caucasian populations (0.7%). This finding is interesting given a report of association of ALDH2 with gout in Japanese individuals [41]. The ALDH2 allele that increased the risk of gout also correlated with increased production of the urate precursor xanthine upon consumption of alcohol [41], giving some insight into the mechanism whereby alcohol increases the risk of gout [42]. As an aside, the Sulem et al. study reported a relatively small OR for SLC2A9 (OR: 1.4) [40], a considerably weaker effect than in other studies [17]. Part of the reason for this could be owing to ascertainment, which included the use of antigout medications, known

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Merriman & McKinney to result in inclusion of nongout cases [43]. In addition, the average age of the Sulem et al. gout sample set was approximately 80 years [40], a group in which there would be expected to be a lesser contribution from genes to gout onset than in a younger case sample set.

Association of serum urate genes with neurological disease The concentration of urate in serum and cerebrospinal fluid is positively correlated [44]. Low levels of urate are a risk factor for Parkinson’s and Alzheimer’s disease [44,45]. Why this is the case is unclear – hypotheses include urate as a neurostimulant, neuroprotection via the activities of urate as an antioxidant and beneficial urate-mediated hypertensive effects [46]. Genetic association between the urate-lowering allele of SLC2A9 and a lower age of onset of Parkinson’s disease [47], and between SLC2A9 and Alzheimer’s disease [48] suggest a causative role for serum urate levels in these neurological conditions. Intriguingly, genetic variation in PDZK1 has been associated with autism [49]. Certainly, genetics should continue to be a useful tool in understanding the etiological role of hypouricemia in neurological disorders. Genetics of acute gout in the presence of hyperuricemia Although hyperuricemia is generally regarded as a necessary prerequisite for gout, epidemiological studies suggest that only between 10 and 20% of those with hyperuricemia develop gout [3]. There are a number of factors that are likely to be involved, for example formation of MSU crystals within joints can be precipitated by local temperature, pH [50] and presence of debris that may act as nucleation centers [51]. However, it is also likely that variations in genes of the innate immune system contribute to the development of gout. MSU crystals play an important role in the immune clearance of dead cells [52]; it is now recognized that gout is an autoinflammatory disorder [53], and functional studies have identified several pathways of the innate immune system that are activated by urate (reviewed in [54]). Therefore, it is likely that precipitated MSU triggers an ‘over-reaction’ by the surveillance mechanism in susceptible individuals, leading to the development of gout. Although there have been several small studies (10 g/dl [3]. Asymptomatic hyperuricemia is not a disease, but rather the underlying factor that can predispose to gout. Sustained hyperuricemia is essential for the development of gout; however, most patients with hyperuricemia will never develop gout. Ultrasonography may be useful in detecting gout in people with hyperuricemia. Puig et  al. reported that 34% of their asymptomatic hyperuricemic individuals had findings suggestive of tophaceous deposits [4]. Pineda et al. studied a larger cohort in a controlled fashion [5]. Ultrasonography changes (the double contour sign and tophi) suggestive of gouty arthritis were found in 25% of hyperuricemic individuals. Tendinous infiltrations of tophaceous material were also observed. These changes were found exclusively in the hyperuricemic individuals but not in their control group of normouricemic individuals. The main limitation of both studies was that participants who were thought to have signs suggestive of gout on ultrasonography did not have a proven diagnosis of MSU crystals; therefore, a definite diagnosis of gout was not established. It is unknown whether ultrasonography evidence suggestive of gout may serve as a noninvasive means to diagnose gout in hyperuricemic individuals who have yet to develop symptomatic gout.

Acute gout The initial manifestation of gout is usually an acute gout attack. The severe pain and agony of the acute gout attack has been the subject of powerful medical recordings and folklore throughout history. Aretaeus, the Greek physician of the second century, stated that “no other pain is more severe than this, not iron screws, nor cords, not the wound of a

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Clinical features of gout dagger, nor burning fire” [6]. Sydney Smith, Box 5.1. Clinical stages in gout. an English essayist, is quoted in 1840 as ƒƒ Asymptomatic hyperuricemia saying that acute gout was “like walking on ƒƒ Acute gout my eyeballs” [7] and in his classic description ƒƒ Intercritical gout of the onset of acute gout, Thomas ƒƒ Chronic tophaceous gout Sydenham, a long sufferer from gout, wrote in London (UK) in 1683: “the victim goes to bed and sleeps quietly. About two in the morning he is awakened by a pain in the great toe; rarely in the heel, ankle or instep. The pain resembles that of a dislocated bone... [It] becomes so exquisitely painful as not to endure the weight of clothes nor the shaking of the room by a person walking in” [8]. Acute gout is characterized by an abrupt onset of severe pain and swelling. Maximal inflammation typically occurs within 4–12 h. The pain is described as the worst pain that the person has ever endured. The first attack often begins at night and wakes the patient up from sleep. It has been suggested that there is a stable level of urate in the joint fluid and that during rest at night the water is absorbed more rapidly than the urate, increasing the concentration of urate or MSU crystals in the joint, which precipitates attacks [9]. During an acute attack (Figure 5.1) the patient endures exquisite pain, which is associated with warmth, redness, swelling and decreased range of motion of the affected joint or joints. Acute attacks last from several hours to several weeks. Typically, the initial acute attacks resolve within 3–10 days in the absence of pharmacologic therapy in the early stages of the disease [10], whereas, subsequent attacks may be more prolonged [10,11] . Fever with temperatures as high as 104°F is not unusual in severe acute gout and may lead to suspicion of an underlying infection. Systemic symptoms and signs of fatigue, fever and chills may accompany acute arthritis. Initial gout attacks most commonly affect the lower extremity joints and are usually monoarticular in men. An acute attack at the first metatarsophalangeal joint (MTPj), known as podagra, is the most common site of an acute attack. MTPj synovitis occurs in 60–80% of patients with gout, and 50% of patients experience their first acute flare in this joint. A study of 615 patients with acute gout found MTPj synovitis in 60% of patients and involvement of bilateral MTPj in 27 cases (4%) [12]. The instep, ankle, heel, knee and hand were affected in the remainder of cases [12,13]. Hips, spine, sacroiliac joints, sternoclavicualr joints and mandibular joints are infrequently affected. Gout can also occur in bursae and tendons. Acute gout in women follows a different pattern. Very few women start with acute podagra (first MTPj arthritis). The most

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Schlesinger Figure 5.1. Acute gout dorsum in the right hand.

common presentation is acute oligopolyarticular gout, especially of the hands, tarsal joints, knees and ankles [14]. Following an initial gout attack, it is estimated that 60% will experience a further attack within 1 year and 80% within 3 years [15]. With subsequent attacks and increasing duration of gout, the frequency of polyarticular attacks increase and there is more frequent involvement of the upper extremities [15,16].

Local trauma and alcohol binges, overeating or fasting have been implicated as factors that precipitate acute gout attacks. Use of diuretics may also increase the risk of gout attacks. In the hospital setting, acute gout attacks often occur postoperatively or are associated with severe acute medical illnesses. Changes in the body’s total uric acid pool can also precipitate an attack, as homeostatic mechanisms mobilize deposited MSU crystals. This is commonly seen in patients newly initiated on urateThe hand is swollen, red, warm and tender to touch. lowering therapy and can be mitigated by slowly titrating the dose upward and adding concomitant prophylactic therapy, such as NSAIDs or colchicine [17,18]. Finally, seasonal factors, such as increased attacks of gout in the spring, have been noted to relate to acute attacks [19].

Intercritical gout After resolution of the acute gout attack, the patient is in the intercritical stage. This period between attacks is referred to as ‘intercritical gout.’ Thus, gout seems to be clinically inactive. Although, clinically, the disease seems quiescent, hyperuricemia is still present and MSU crystal formation and deposition may continue, as well as ongoing subclinical inflammation [1]. Chronic tophaceous gout Chronic tophaceous gout usually develops after ≥10  years of acute intermittent gout, although patients have rarely presented with tophi as their initial manifestation of the disease [20]. However, microtophi are

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Clinical features of gout suspected to form in the early stages of gout and have been documented in patients with hyperuricemia [4,5]. Tophi appear as firm swellings. They may appear at any site. The most common sites for tophi to appear are in the digits of the hands and feet (Figure 5.2), as well as in the olecranon bursa. Tophi may be associated with a destructive deforming arthritis and may ulcerate, in which case secondary infection may be a problem. Tophi of the helix or antihelix of the ear are classical but less common. Tophi have been reported in the eye [21], carpal tunnel [22] and heart valves [23]. In these situations the diagnosis is often unsuspected until surgery. In women there is a tendency to develop tophaceous deposits on Heberden’s and Buchard’s nodes (hard bony enlargements of the small joints of the fingers seen in osteoarthritis), sometimes with minimal inflammation [24]. As clinicians treating patients with gout, it is our job to prevent the development of chronic tophaceous gout. Over time, even in the absence of attacks or with only few attacks, MSU crystal deposition and inflammation may lead to the development of clinically evident joint damage and erosions. Figure 5.2. Tophaceous gout bilateral feet.

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Schlesinger Financial & competing interests disclosure N Schlesinger reports having received a grant, travel expenses and payment for advisory board membership from Novartis Pharma, payment for advisory board membership and educational presentations from Takeda and Savient, and payment for advisory board membership from Savient, URL Pharma, and Enzyme Rx. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ Most hyperuricemics will never develop gout. ƒƒ Acute gout is characterized by rapid onset and build-up of pain with a resolution within days to several weeks. ƒƒ Most patients experience recurrent acute gout attacks. The frequency of attacks varies between patients. ƒƒ The patient may be asymptomatic during the intercritical period, but inflammation and monosodium urate formation and deposition may continue. ƒƒ Over several years, chronic tophaceous gout may ensue. ƒƒ Tophi are most commonly observed as subcutaneous deposits overlying joints and around the joints, but have been reported in other sites, such as the eye, carpal tunnel and heart valves. ƒƒ As clinicians treating patients with gout, it is our job to prevent the development of chronic tophaceous gout.

References 1

Schlesinger N, Thiele RG. The pathogenesis of bone erosions in gouty arthritis. Ann. Rheum. Dis. 69(11), 1907–1912 (2010).

2

Campion EW, Glynn RJ, DeLabry LO. Asymptomatic hyperuricemia: risks and consequences in the Normative Aging Study. Am. J. Med. 82, 421–426 (1987).

3

Agudelo C, Wise CM. Crystalassociated arthritis. Clin. Geriatr. Med. 14, 495–513 (1998).

4

Puig JG, de Miguel E, Castillo MC, Rocha AL, Martínez MA, Torres RJ.

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Asymptomatic hyperuricemia: impact of ultrasonography. Nucleosides Nucleotides Nucleic Acids 27, 592–595 (2008).

7

Lady Holland. A Memoir of Reverend Sydney Smith. Lady Holland (Ed.). Longmans, Green & Co., London, UK (1855).

5

Pineda C, Amezcua-Guerra LM, Solano C et al. Joint and tendon subclinical involvement suggestive of gouty arthritis in asympto­matic hyperuricemia: an ultrasound controlled study. Arthritis Res. Ther. 13, R4 (2011).

8

Syndenham T. Selected Works of Thomas Syndenham MD with a Short Biography and Explanatory Notes. Comrie JD (Ed.). John Bale, Sons and Danielsson, London, UK (1922).

Weeden RP. Poison in the Pot: the Legacy of Lead. Southern Illinois University Press: Carbondale and Edwardsville, IL, USA, 83, (1984).

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6

Simkin PA. The pathogenesis of podagra. Ann. Intern. Med. 86, 230–233 (1977).

10 Schlesinger N. Diagnosis of

gout. Minerva Med. 98(6), 759–767 (2007).

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Clinical features of gout 11 Schlesinger N. Diagnosis of

gout: clinical, laboratory, and radiologic findings. Am. J. Manag. Care 11(Suppl. 15), S443–S450 (2005).

16 Lawry GV 2nd, Fan PT,

12 Scudamore C. A Treatise on

the Nature and Cure of Gout (4th Edition). Kessinger Publishing, PA, USA (1923).

13 Grahame R, Scott JT. Clinical

survey of 354 patients with gout. Ann. Rheum. Dis. 29, 461–468 (1970).

17

A comparison of gout in men and women: a 10-year experience. S. Afr. Med. J. 70, 721–723 (1986).

Currie WJ. The gout patient in general practice. Rheumatol. Rehabil. 17(4), 205–217 (1987).

22 Champion D. Gouty

acute and chronic gouty arthritis: present state-of-theart. Drugs 64(21), 2399–2416 (2004).

19 Schlesinger N, Baker DG,

Beutler AM, Hoffman BI, Schumacher HR Jr. Acute gouty arthritis is seasonal. J. Rheumatol. 25(2), 342–344 (1998).

15 Ferraz MB, O’Brien B. A cost

effectiveness analysis of urate lowering drugs in nontophaceous recurrent gouty arthritis. J. Rheumatol. 22(5), 908–914 (1995).

21 Martinez-Cordero E, Barriera-

18 Schlesinger N. Management of

14 Meyers OL, Montegudo FSE.

20 Wernick R, Winkler C,

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of six cases and review of the literature. Arch. Intern. Med. 152(4), 873–876 (1992).

Bluestone R. Polyarticular versus monoarticular gout: a prospective, comparative analysis of clinical features. Medicine (Baltimore) 67(5), 335–343 (1988).

Campbell S. Tophi as the initial manifestation of gout: report

Mercado E, Katona G. Eye tophi deposition in gout. J. Rheumatol. 13(2), 471–473 (1986). tenosynovitis and the carpal tunnel syndrome. Med. J. Aust. 1(20), 1030–1032 (1969).

23 Scalapino JN, Edwards WD,

Steckelberg JM, Wooton RS, Callahan JA, Ginsberg WW. Mitral stenosis associated with valvular tophi. Mayo Clin. Proc. 59(7), 509–512 (1984).

24 Lally EV, Ho G Jr, Kaplan SR.

The clinical spectrum of gouty arthritis in women. Arch. Intern. Med. 146(11), 2221 (1986).

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About the Authors Eliseo Pascual Eliseo Pascual is Professor of Medicine and Head of the Rheumatology Section at the Hospital General Universitario de Alicante (Spain) and the Universidad Miguel Hernández (Alicante, Spain). His interest in gout started during his training at the Hospital of the University of Pennsylvania (USA) under Joseph Hollander and Ralph Schumacher. He has continued routinely looking at all synovial fluids obtained from arthritis of uncertain etiology, and in the last 12 years has organized and chaired the workshop on crystal analysis at the European League Against Rheumatism Congresses, having written papers and reviews on the subject.

Mariano Andrés Mariano Andrés works as a clinical associate in the Rheumatology Section of Hospital General Universitario de Alicante. He graduated in 2006 from medical school, and completed his training in rheumatology in 2011 under supervision of Eliseo Pascual. His current fields of interest are crystal-related arthritis and vasculitides.

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Chapter

6 Synovial fluid analysis and crystal identification

Preparation of the sample 81 The microscope

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Some practical tips

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Other characteristics of SF in crystal arthritis

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Other crystals in SF

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Eliseo Pascual & Mariano Andrés The search for and identification of crystals is mandatory when synovial fluid can be obtained from an arthritis that has not been diagnosed with certainty, as this provides the definitive diagnosis for crystal-related arthritis, which, aside from their most characteristic presentations, can clinically be quite varied and consistent with other joint diseases of less certain diagnosis. The gross and microscopic analysis of synovial fluid allows differentiation between inflammatory and noninflammatory entities, but no further diagnosis can be achieved, except for crystal arthritis and infections. Gout and calcium pyrophosphate crystal arthritides are common diseases; apart from apatite crystals, their investigation has unclear usefulness in clinical practice, other crystals described in synovial fluid samples are of anecdotal importance. Therefore, this chapter will be centered on monosodium urate and calcium pyrophosphate crystal arthritis, with a short focus on other rare crystals of anecdotal practical relevance.

doi:10.2217/EBO.12.222

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Pascual & Andrés Since its initial description, synovial fluid (SF) analysis for crystals has received little critical attention and the technique remains essentially unchanged. Crystal identification in SF is included in the core curriculae in rheumatology, both by the American College of Rheumatology [101] and by the European Union of Medical Specialists [102]; despite this, it has been relegated, and only a minority of rheumatologists routinely base the diagnosis of crystal arthritis on crystal identification [1]. Interestingly, explanations such as crystal analysis being inconvenient or time consuming are often given to justify the diagnosis of crystal arthritis on an inaccurate clinical approach (at least in less characteristic presentations); indeed crystal arthritides are quite often not taken seriously [2]. The absence of interest in crystal arthritis that may be perceived by trainees from their senior colleagues seriously hampers their interest and training in the procedure.

The identification of monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals in synovial fluids or tissue samples allows a definitive diagnosis of gout and calcium pyrophosphate deposition disease, respectively.

Crystal identification is a simple procedure requiring only a microscope fitted with polarized filters and a first-order red compensator filter and after training the results of crystal analysis are consistent [3]. We cannot think of any other procedure that is so immediate and allows an unequivocal etiologic diagnosis in any other disease. Crystal analysis can be taken as a bedside procedure allowing an immediate and definitive diagnosis and treatment of crystal arthritis that is not possible for any other arthritis [4]. Finally, it may be felt that SF analysis is impossible without the fully equipped compensated polarized microscope – the use of which is more complex than that of the simple microscope – especially for those less familiar with the use of the microscope, but the ordinary microscope already offers a reasonably good identification of both monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals [5] , and the same microscope fitted with polarized filters (available in most pathology departments) offers a very reasonable kit that allows a fruitful start in crystal analysis in SF. MSU and CPP crystals are regularly found in SF samples obtained from inflamed joints of their related diseases. Furthermore, MSU crystals are regularly found in previously inflamed asymptomatic gouty joints of patients untreated with serum uric acidlowering drugs [6,7], allowing the diagnosis during intercritical periods. Such a finding Red compensator filter: helps to determine is not surprising since ultrasound studies [8,9] whether the light passing through an axe of the and arthroscopic observations [10] show crystal suffered more or less retardation in its that MSU crystals deposit at the surface of wavelength, establishing the type of birefringence (positive or negative) that crystals have – and helping the joint cartilage, directly bathed by SF and to distinguish MSU from CPP crystals.

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Synovial fluid analysis & crystal identification Synovial fluid crystal analysis by compensated in an area of pressure and friction during polarized light microscopy is the gold standard joint movements. MSU crystals disappear for MSU and CPP crystal identification. However, after a long enough treatment with serum simple polarized and ordinary light microscopy is most uric acid-lowering drugs [11]. Needling a often sufficient for precise diagnosis. tophus most often brings urate crystals, usually in small amounts that stay inside the needle; therefore, air has to be flushed through it with a syringe to get them out, which can then be observed with a microscope. CPP crystals are also found in SF samples obtained from asymptomatic joints [12]. In pathological preparations, MSU crystals may be absent since they are dissolved by formalin during the necessary fixation process [13]. Alcohol fixation has been recommended; the authors have good experience with stained frozen sections.

Preparation of the sample SF is examined fresh; no fixation or staining is needed. Fresh samples are necessary – especially for CPP – to avoid decay of the cells, which may make crystal analysis difficult. The samples can be kept at 4°C and, in general, examination after 24–36  h is adequate. If examination is not immediate, clotting of highly inflammatory samples can be avoided by adding a drop of heparin. A small drop of the fluid is placed on a glass slide and covered with a cover slip (larger drops result in less-appropriate, thicker preparations). The microscope Most regular microscopes used for bright-field microscopy can be fitted with appropriate filters allowing simple polarized and compensated polarized microscopy. When asked about a polarized microscope, manufacturers may offer a geological polarized microscope, a more expensive tool that grades rotating stage, allowing the individual to determine the angles of extinction (position where the crystal loses its birefringence) of the different crystals, but unnecessary to distinguish MSU from CPP crystals, and MSU and CPP crystals from artifacts (a frequent cause of hesitation and trouble for beginners and seldom for experienced analysts). A 200–400× lens is ideal for MSU crystal detection and identification; a 600× lens is better for CPP crystals, identification of which often relies on shape; this is – for this purpose – the preferred lens of the authors. Starting with the simplest tool, and building up in complexity, crystal analysis can be approached as follows: Bright-field microscopy shows the crystals well, allowing reasonable detection (if

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Birefringence: ability of crystals to decompose the light beam when it passes through them.

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Pascual & Andrés Figure 6.1. Monosodium urate crystals seen at 400×. A

B

C

λ

Two cells are seen, each contains a needle-shaped crystal. (A) Bright-field (ordinary) microscope shows them by shape. (B) Simple polarized microscope shows strong birefringence in both crystals. (C) Negative birefringence (yellow if parallel and blue if perpendicular to the compensator axis marked with a λ) allows definitive identification according to current standards. The arrow marked by λ indicates the direction of the axis of the compensator.

there are crystals, they are seen) and identification as MSU and CPP crystals by their shape [5]. Especially in bright-field microscopy, the height of the microscope condenser must be carefully regulated to obtain the best contrast and vision, and the condenser or the microscope diaphragm should not be closed. All MSU crystals are needle shaped, although their size can vary (Figures  6.1A, 6.2A, 6.3A & 6.4A); crystals obtained by needling a tophus are larger than those obtained in a SF sample. Crystals are seen intra- and extra-cellularly, and although initially it was felt that intracellular crystals meant joint inflammation, these are common in SF samples obtained from asymptomatic gouty joints (where indeed a low grade of subclinical inflammation does exist) [14]. In the authors’ opinion, bright-field microscopy is the most appropriate method for detection and identification of CPP crystals. CPP polymorphic crystals may pose more Figure 6.2. Monosodium urate crystals seen at 600×. A

B

C

λ

(A) Bright field-microscope shows them by shape (regulating the microscope condenser height allows the individual to determine the best contrast and vision). (B) Simple polarized microscope shows strong birefringence in both crystals. (C) Negative birefringence allows definitive identification according to current standards. The arrow marked by λ indicates the direction of the axis of the compensator.

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Synovial fluid analysis & crystal identification Figure 6.3. Small monosodium urate crystals seen at 400×. A

B

C

λ

(A) Bright-field microscopy does not allow clear vision of the crystal (under the microscope the crystal was visible – better distinguished by focusing up and down through the cell with the thin focusing knob). (B) Simple polarized microscope shows strong birefringence; a second faintly shining crystal is distinguished in the upper part of the same cell. (C) The negative birefringence of the crystal definitively identifies it as monosodium urate (MSU) crystal. These figures illustrate that for crystal detection (if there are MSU crystals, they will be seen) simple polarized microscopy is the best technique; small MSU crystals easily pass undetected by the bright-field microscope. The arrow marked by λ indicates the direction of the axis of the compensator.

difficulties. Under bright-field microscopy, crystal shape varies from easily recognizable rhombi and parallelepipeds or rectangles to rod-looking very long rectangles and finally needles that look under the bright-field microscope like MSU crystals (Figures 6.5A, 6.6A, 6.7A, 6.8A, 6.9A, 6.10A & 6.11A). When these MSU-looking needles are the first finding in the analysis, the search must continue until finding clear CPP-looking crystals – in which case all are most likely CPP, or it becomes clear that all crystals are needle shaped, thus very likely MSU. The bright-field microscope is not a proper tool for identifying those very rare SF samples that contain both CPP and MSU (see discussion at the end of the chapter). CPP crystals frequently show a less regular shape, usually because of their position; in Figure 6.4. Abundant monosodium urate crystals seen at 600×. A

B

C

λ

The presence of large crystals is not unusual in synovial fluid of gout of long duration (and such large crystals are obtained most often from needling a tophus). Note the absence of cells, highly suggestive that the fluid was obtained from a clinically uninflamed joint. (A) Bright-field microscope, (B) simple polarized microscope and (C) compensated polarized microscope. The arrow marked by λ indicates the direction of the axis of the compensator.

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Pascual & Andrés Figure 6.5. Rectangular calcium pyrophosphate crystal seen at 400×. A

B

C

λ

(A) Bright-field microscope shows it well by its shape, allowing identification. (B) Observed under simple ­ olarized light, the crystal shows some birefringence – clearly fainter that than of monosodium urate under p the microscope. (C) The crystal shows positive birefringence (yellow when parallel to the compensator axis (λ), allowing definitive identification according to current standards. The bright-field microscope is the best tool for detecting calcium pyrophosphate crystals and allows accurate identification by shape. The arrow marked by λ indicates the direction of the axis of the compensator.

fresh preparations cell frequently move, and as they move, CPP crystals contained in them change shape often from characteristic to more irregular shapes, and observing this is a useful learning experience. Very small intracellular crystals are common; MSU always as tiny needles, but CPP may show as tiny needles or very small rhombi or just refractive cell inclusions that may present an angle in them (Figure 6.12A). These small fragments are not sufficient for crystal identification but they show that if the search continues, more characteristic crystals will most likely be found. CPP crystals are frequently found inside a vacuole, and this is not the case for MSU crystals. These smaller crystals are much better seen under a greater magnification with the 1000× oil lens. A 600× lens is Figure 6.6. Calcium pyrophosphate crystals seen at 600×. A

B

C

λ

(A) Bright-field microscope shows well by its shape an intracellular rectangular crystal; a small needle-shaped crystal is seen attached to the left side of the cell. (B) The large crystal shows very faint birefringence under the simple polarized microscope. (C) The compensated polarized microscope shows the crystal with positive birefringence – blue when parallel to the compensator axis. The arrow marked by λ indicates the direction of the axis of the compensator.

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Synovial fluid analysis & crystal identification Figure 6.7. Calcium pyrophosphate crystals seen at 400×. A

B

C

λ

(A) Under the bright-field microscope, the crystals are well detected and identified by their shape. (B) Under the simple polarized microscope, some crystals show faint birefringence; others are not distinguished. (C) Compensated polarized microscopy allows definitive identification – according to current standards – by their positive birefringence. The arrow marked by λ indicates the direction of the axis of the compensator.

particularly appropriate when searching for CPP crystals. As a teaching exercise, observing CPP crystals under a 1000× oil lens helps in familiarizing with their shape. The ordinary bright-field microscope appears to be the best tool for CPP crystal detection. Simple polarized microscopy allows the detection of crystals by their birefringence. It requires the microscope to be fitted with two polarization filters, one below (polarizer) and one above (analyzer) the stage. After passing the first, crystal light vibrates in only one plane; if the second filter is rotated so its axis is placed perpendicular to the axis of the first filter, no light passes through and the microscope field becomes dark. This polarized light passing though the birefringent crystals is decomposed and one of its components emerges parallel to the axis of the second filter and passes through it, so it is seen in the shining in the dark microscope field [103]. This facilitates crystal detection, especially that of the strongly birefringent MSU crystals. Besides crystals, other materials – and frequently artifacts – also show birefringence. All needle-shaped MSU crystals show brilliant birefringence and clearly shine in the dark field (Figures 6.1B, 6.2B, 6.3B & 6.4B). This is already noticeable at 200× (which allows the examination of a larger microscope field and faster detection). All crystals show a closely intense birefringence but, occasionally, MSU crystals may lack it. This occurs if a crystal axis is positioned parallel to one of the axes of the analyzer or polarizer, and also with some very small crystals – although other small crystals may shine intensely. The simple polarized Polarizer: optical filter that only passes light microscope appears to be the best tool for that is vibrating parallel to its axis. When two polarizer filters are placed so that their axes are MSU crystal detection, showing crystals that perpendicular, light passing the first filter is blocked are less apparent after bright-field by the second, not reaching the ocular and resulting observation (Figures 6.3A & 6.3B). in a dark field.

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Pascual & Andrés Figure 6.8. Calcium pyrophosphate crystals seen at 600×. A

B

(A) Bright-field microscopy allows the distinction of both rectangular and needle-shaped crystals. (B) Under simple polarized light, most crystals do not shine or do it very faintly. Note the faint birefringent of the needle-shaped crystals, which allows the individual to distinguish them from the highly birefringent monosodium urate crystals.

On the other hand, only approximately a fifth of CPP crystals show any birefringence that is fainter than that of the brilliant ones of MSU crystals (Figures 6.8A, 6.8B, 6.11A & 6.11B)  [15]. So, if searched under simple polarized microscopy, CPP crystals are easily missed. It is the author’s feeling that acicular CPP crystals seldom show birefringence and if they do, it is much fainter than that of MSU crystals, and this appears to be an important differentiating element if compensated polarized microscopy is unavailable. Compensated polarized microscopy remains the standard for crystal identification; the technique is more complex and for those in the learning Figure 6.9. Rhomboidal calcium pyrophosphate crystal seen at 600×. A

B

C

λ

A small needle-shaped crystal is seen at the right of the cell. (A) Under the bright-field microscope, the crystals are well seen and the rhomboidal shape allows identification. (B) Under simple polarized light, the rhomboidal crystal is clearly birefringent; the smaller crystal is not distinguished. Compensated polarized ­microscopy shows blue the rhomboidal crystal, but because of its shape it cannot be oriented in relation to the compensator axis. The small needle-shaped crystal shows faint, positive birefringence – yellow ­perpendicular to λ. The arrow marked by λ indicates the direction of the axis of the compensator.

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Synovial fluid analysis & crystal identification Figure 6.10. Large rectangular calcium pyrophosphate crystal seen at 600×. A

B

C

λ

(A) Bright-field microscope. (B) Simple polarized microscope showing very modest birefringence. (C) Compensated polarized microscope showing positive birefringence. The arrow marked by λ indicates the direction of the axis of the compensator.

process it appears reasonable to approach it after becoming acquainted with the crystals with the ordinary and simple polarized microscopes. It adds to the previous system a first-order red compensator (retardation plate; its axis usually marked by a l and an arrow), which helps to determine the amount of retardation in the wavelength of the compound ray emerging from the long dimension of the birefringent crystal. When this ray is less retarded, it shows yellow when parallel to the compensator axis and blue if perpendicular to it, and the crystal is said to have negative birefringence. When the vibration of the slower ray is parallel to the long dimension of the crystal, it shows blue when parallel to the compensator axis and yellow if perpendicular to it, and it is said to have positive Figure 6.11. Aggregate of cells containing large numbers of calcium pyrophosphate crystals, seen at 600×. A

B

C

λ

(A) The bright-field microscope shows calcium pyrophosphate crystals of different shapes. (B) Simple polarized microscope shows very variable birefringence; only very few show strong birefringence, and many do not show any. (C) Polarized compensated microscope shows that: some crystals not showing birefringence under the simple polarized microscope do not change color to yellow or blue; and very few rectangular/parallel epipedic crystals show color and can be oriented in relation to λ, thus allowing identification according to current standards. The arrow marked by λ indicates the direction of the axis of the compensator.

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Pascual & Andrés Figure 6.12. Very small calcium pyrophosphate crystals. A

B

C

(A) 600×; small irregularly shaped refractile inclusion. (B) 1000× oil lens; two small irregularly shaped inclusions. The finding of these irregular, refractile inclusions, often with a single 90° corner, do not allow any diagnosis, but often herald the finding of characteristic calcium pyrophosphate (CPP) crystals if the search is pursued. (C) 1000× oil lens; shows a small intracellular acicular crystal. In this case, the absence of bi­refringence suggested that it is CPP. Further search resulted in the finding of characteristic CPP crystals. These findings that are insufficient for diagnosis but suggestive should prompt the continuation of the search for characteristic diagnostic crystals.

birefringence [16,104]. Compensated polarized microscopy helps in the distinction of strongly negatively birefringence of MSU crystals; these crystals are easily positioned in relation to the compensator axis and easily identified (Figures 6.1C–6.4C). CPP crystals show a weakly positive birefringence and, due to the variability in shape, only rectangular and parallel epipedic crystals – and those showing some birefringence under the simple polarized microscope – are easily identified, but rhombi cannot be oriented in relation to the compensator axis and many rod or needleshaped crystals do not show clear birefringence (Figures 6.5C, 6.6C, 6.7C, 6.9C, 6.10C, 6.11C & 6.12C). Although analysis with this filter set (compensated polarized) remains the standard tool for definitive MSU and CPP crystal distinction, the crystal nature is already evident in most occasions based on shape under ordinary microscope and intensity of birefringence under simple polarized light for needle-shaped crystals. Compensated polarized microscopy is most useful in the identification of very occasional needle-like artefacts, or when MSU and CPP crystals coincide in the same SF; this occurrence appears sporadic and no report on its clinical consequences has been published. A recent report examining gout SF after cytocentrifugation has found a few CPP crystals in gouty joints that also shows moderate-to-severe osteoarthritis, and are possibly related to it [17]. Crystal shape and birefringence (strongly negative in MSU crystals and weakly positive in calcium pyrophosphate crystals) help distinguish between MSU and calcium pyrophosphate crystals.

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Some practical tips For the beginner, it is practical to approach crystal analysis in two separate steps: crystal detection to ascertain whether

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Synovial fluid analysis & crystal identification The learning process can be started with a there are MSU or CPP crystals (or not) in the bright-field microscope, and looking at synovial SF sample being analyzed – it must be kept fluid samples of known origin to detect and identify in mind that when infection occurs in a the crystals by shape. A simple polarized microscope crystal-containing joint, crystals will be (usually available at pathology departments) allows found in the SF; and crystal identification, distinction by intensity/absence of birefringence. in which the detected crystals will be Crystal identification by microscopy continues to be properly identified as MSU or CPP [14,18]. It underutilized in clinical practice, despite being a simple, rapid and reliable diagnostic tool. must be noted that CPP crystals pose more difficulties. For MSU crystals, book descriptions fit quite well with the very large majority of crystals, all needle shaped and almost all strongly birefringent under simple polarized light; but for CPP crystals, books and reviews tend to show the most characteristic crystals – and those are expected to be easily identified by the trainee. However, as mentioned above, CPP crystals are polymorphic and many do not show birefringence, and those doing it show a faint shine in comparison to MSU. Many CPP crystals (those small, not properly oriented in the microscope field and many needle- or rod-shaped, or less regular) may be difficult until the analyst gains in experience and CPP crystals become familiar in all their appearances. Although to beginners the process appears to require careful analysis, experienced analysts most often recognize the crystal type at first glance.

The objective of the learning process is to become acquainted with the appearance of the crystals under the different types of filtration, and to do so, trainees should examine SF samples of known origin, detect and identify the crystals and spend time becoming familiar with their appearance so that they will be able to distinguish them easily afterwards. A common error is to be prevented from ever looking at crystals owing to the concept that crystal analysis is only possible by means of a compensated polarized microscope. In this chapter, we outline the possibility of doing it starting with an ordinary, bright-field microscope. To aid in the process, most figures show the same field with bright field and with crossed polarized filters and the same after adding a first-order red compensator. By doing so, we are trying to encourage those motivated to start their learning process. The confusing capacity of artefacts of different origins – simple dust being a common one – is especially problematic for the beginner, who should be aware of them, and become acquainted with their appearance. Leaving a glass slide on a table for a couple of days and looking at it afterwards under polarized microscopy may provide an idea of their confusing potential. The effort needed to detect the crystals, if present, or to ascertain their absence has received little attention. Different lengths of time of observation have been proposed, but different analysts observe at different speeds. It

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Pascual & Andrés Figure 6.13. Apatite crystal agglomerates, bright-field microscope, 600×.

Probably due to the rupture of a calcification inside a joint – or a bursa – that may rarely result in an acute arthritis from which a cloudy (often very cloudy) fluid is obtained and these apatite clumps are seen. Radiological calcification or its finding by sonography may provide an additional clue for the right diagnosis.

may be more practical to examine a number of 400× microscope fields – 30 to 60 – before determining that there are no crystals. For gout, it is practical when the diagnosis appears possible and no crystals are found to centrifuge the SF sample and examine the pellet, where crystals concentrate. MSU crystals do not occur outside of gout, so a simple crystal has to be taken as diagnostic – although a diagnosis based on such a limited finding must be taken as provisional and confirmed on a later occasion or in a SF sample obtained from a different joint. A particular problem results from the finding of an occasional CPP crystal. This occurs in osteoarthritis SFs, and it has been reported after meniscectomy [19] and in osteoarthritis occurring after joint dystrophies. It remains unclear how to interpret the finding of these occasional CPP crystals and how many of them it is necessary to make a sound diagnosis of CPP arthopathy.

When infection occurs in joint-containing crystals, they easily show in the SF and infection can be missed or not considered. When clinical findings suggest this possibility, it is mandatory to culture a sample of the fluid.

Other characteristics of SF in crystal arthritis SF samples obtained from joints at the time of an attack of gout or CPP arthritis are inflammatory, thus the appearance is cloudy. Inflammation in these joints can be high and cell counts above 50,000 cells/µl suggest infection [20], especially if smaller joints are affected, as cellularity in smaller joints tends to be higher [21]. Some more chronic effusions can show quite transparent SF samples and, on occasions, faint cloudiness originates from a high content of crystals – mostly in gout – with few cells. SF can be chalk white when it contains a high concentration of MSU crystals and no other elements. Finally, on occasions, white speckles can be seen floating in inflammatory – or even quite noninflammatory – SF samples, and those visualized under the polarized microscope show that they are composed of MSU crystals.

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Synovial fluid analysis & crystal identification Figure 6.14. Cholesterol crystals, seen at 600×. A

B

C

λ

(A) Bright-field microscope; (B) simple polarized light microscope; (C) compensated polarized microscope. These large plaques frequently with a notched corner can be seen in chronic effusions of different diseases. The arrow marked by λ indicates the direction of the axis of the compensator.

Other crystals in SF Basic calcium phosphate crystals – the most common is apatite – can be detected in osteoarthritic joints or rapidly destructive arthritis (e.g., Milwau­kee’s shoulder). Owing to their small size, basic calcium phosphate crystals are undetectable with light microscopy, although can be suspected when amorphous aggregates are seen by bright-field microscopy (Figure 6.13). Staining with alizarin red helps to detect apatite crystals, but as it stains any calcium salt, other crystals (i.e., CPP) also show weakly. Outside the research setting, the convenience of routinely searching for apatite crystals remains undefined. Cholesterol crystals are typically found in long-standing effusions of joints [22] , most commonly in rheumatoid arthritis joints, tendon sheet and bursae, but also in synovial samples of other origin, including crystal arthritis. Figure 6.15. Triamcinolone acetonide crystals, seen at 600×. A

B

C

λ

(A) Bright-field microscope shows triamcinolone acetonide crystals, as well as, roundish, irregular, mostly intracellular, inclusions. (B) Simple-polarized microscope shows their high birefringence. (C) Compensated polarized microscope does not add any useful information (other corticosteroid crystals can be elongated, or even needle shaped, and compensated-polarized microscopy may help in distinguishing them from monosodium urate). The arrow marked by λ indicates the direction of the axis of the compensator.

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Pascual & Andrés Cholesterol crystals are large plates with a notched corner, with variable birefringence (Figure 6.14); occasional cholesterol crystals may show as long, curved, needle-like crystals that should be taken for MSU crystals. Their pathogenic potential appears small, if any. Calcium oxalate crystals, previously identified in patients under­going hemodialysis, now are a rarity, with scant reports in peritoneal dialysis [23] or in patients with primary hyperoxaluria [24]. The classical bipyramidal shape is only displayed by a few crystals, while the rest are irregular or rod shaped. Calcium oxalate might depict chondrocalcinosis. The finding of other type of crystals (e.g., hematoidin, lipid ‘maltese cross’ and Charcot–Leyden crystals) is anecdotal. Finally, corticosteroid crystals in SF result from a previous intra-articular injection of a corticosteroid preparation, and the crystals can persist for an undefined period of time. In some patients, a postinjection flare may develop, simulating an infection [25]. Steroid crystals can be seen under light microscopy with different shapes, depending on the corticosteroid preparation injected [26], usually with strong, positive birefringence (Figure 6.15). Financial & competing interests disclosure E Pascual has served in boards for Manarini and Savient, and has given lectures for Menarini. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ Synovial fluid analysis for crystals is the most accurate method to firmly diagnose both gout and calcium pyrophosphate crystal arthritis. The method remains unchanged since the descriptions by McCarty in the 1960s, as the field has received scant critical attention so far. ƒƒ Crystal shape in the bright-field microscope and the presence or absence of birefringence after polarization allows precise crystal identification, rarely requiring compensated polarised microscopy that remains the standard procedure. ƒƒ Other crystals may be found in synovial fluid, but are rare or have an uncertain pathogenic potential. ƒƒ This chapter outlines the crystal identification method, especially from a practical point of view, giving some tips and instructions for beginners.

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Synovial fluid analysis & crystal identification References 1

Amer H, Swan A, Dieppe P. The utilization of synovial fluid analysis in the UK. Rheumatology (Oxf.) 40, 1060–1063 (2001).

2

Pascual E, Sivera F. Why should be gout so poorly treated? Ann. Rheum. Dis. 66, 1269–1270 (2007).

3

Lumbreras B, Pascual E, Frasquet J, González-Salinas J, Rodríguez E, HernándezAguado I. Analysis for crystals in synovial fluid: training of the analysts results in high consistency. Ann. Rheum. Dis. 64, 612–615 (2005).

4

5

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Gatter RA. Gross synovianalysis at the bedside. In: A Practical Handbook of Joint Fluid Analysis. Gatter RA (Ed.). Lea and Febiger, PA, USA, 15–20 (1984). Pascual E, Tovar J, Ruiz MT. The ordinary light micros­ cope: an appropriate tool for the detection and identification of crystals in synovial fluid. Ann. Rheum. Dis. 48, 983–985 (1989). Pascual E. Persistence of monosodium urate crystals and low-grade inflammationin the synovial fluid of patients with untreated gout. Arthritis Rheum. 34(2), 141–145 (1991). Pascual E, Batlle-Gualda E, Martínez A et al. Synovial fluid analysis for diagnosis of intercritical gout. Ann. Intern. Med. 131, 756–759 (1999). Grassi W, Menga G, Pascual E, Filipucci E. ‘Crystal Clear’ – sonographic assessment of gout and calcium pyrophosphate deposition disease. Semin. Arthritis Rheum. 36, 197–202 (2006).

9

Wright SA, Filippucci E, McVeigh C et al. Highresolution ultrasonography of the first metatarsal phalangeal joint in gout: a controlled study. Ann. Rheum. Dis. 66, 859–864 (2007).

10 Baker JF, Synnott KA. Clinical

images: gout revealed on arthroscopy after minor injury. Arthritis Rheum. 62, 895 (2010).

11 Pascual E, Sivera F. The time

required for disappearance of urate crystals from synovial fluid after successful hypouricemic treatment relates to the duration of gout. Ann. Rheum. Dis. 66, 1056–1058 (2007).

12 Martinez-Sanchis A,

Pascual E. Intracellular and extracellular CPPD crystals are a regular feature in synovial fluid from uninflamed joints of patients with CPPD related arthropathy. Ann. Rheum. Dis. 64, 1769–1772 (2005).

13 Simkin PA, Bassett JE, Lee

QP. Not water, but formalin, dissolves urate crystals in tophaceous tissue samples. J. Rheumatol. 21, 2320–2321 (1994).

14 Pascual E, Jovani V. Synovial

fluid analysis. Best Pract. Res. Clin. Rheumatol. 19, 371–386 (2005).

15 Ivorra J, Rosas E, Pascual E.

Most calcium pyrophosphate crystals appear as nonbirefringent. Ann. Rheum. Dis. 58, 582–584 (1999).

16 Phelps P, Steele AD,

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McCarty DJ Jr. Compensated polarized light microscopy. Identification of crystals in

synovial fluids from gout and pseudogout. JAMA 203, 508–512 (1968). 17

Robier C, Neubauer M, Quehenberger F, Rainer F. Coincidence of calcium pyrophosphate and monosodium urate crystals in the synovial fluid of patients with gout determined by the cytocentrifugation technique. Ann. Rheum. Dis. 70, 1163–1164 (2011).

18 Courtney P, Doherty M. Joint

aspiration and injection and synovial fluid analysis. Best Pract. Res. Clin. Rheumatol. 23, 161–192 (2009).

19 Doherty M, Watt I, Dieppe PA.

Localised chondrocalcinosis in post-meniscectomy knees. Lancet 29, 1207–1210 (1982).

20 Frischnecht J, Steigerwald JC.

High synovial fluid white in pseudogout; possible confusion with septic arthritis. Arch. Intern. Med. 135, 298–299 (1975).

21 Pascual Gomez E. Joint size

influences on the leukocyte count of inflammatory synovial fluids. Br. J. Rheumatol. 28, 28–30 (1989).

22 Ettlinger RE, Hunder CG.

Synovial effusions containing cholesterol crystals report of 12 patients and review. Mayo Clin. Proc. 54, 366–374 (1979).

23 Rosenthal A, Ryan LM,

McCarty DJ. Arthritis associated with calcium oxalate crystals in an anephric patient treated with peritoneal dialysis. JAMA 260, 1280–1282 (1988).

24 Verbruggen LA, Bourgain C,

Verbeelen D. Late presentation and microcrystalline arthropathy

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Pascual & Andrés in primary hyperoxaluria. Clin. Exp. Rheumatol. 7, 631–633 (1989). 25 McCarty DJ, Hogan JM.

Inflammatory reaction after intrasynovial injection of microcrystalline adrenocorticosteroid esters. Arthritis Rheum. 7, 359–367 (1964).

26 Kahn CB, Hollander JL,

Schumacher HR. Corticosteroid crystals in synovial fluid. JAMA 211, 807–809 (1970).

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Websites 101 American College of

Rheumatology. Core curriculum outline for rheumatology fellowship programs: a competencybased guide to curriculum development (March 2006). www.rheumatology. org/education/training/cco. pdf

102 UEMS, Section of

Rheumatology: core curriculum for specialist training.

www.eular.org/ myUploadData/files/UEMS_ Rheumatology_Specialist_ Core_Curriculum_2003.pdf 103 Olympus Microscopy Resource

Center. Polarized-light microscopy. www.olympusmicro.com/ primer/techniques/polarized/ polarizedhome.html

104 Nikon MicroscopyU. The

source for microscopy education. www.microscopyu.com/ articles/polarized/index.html

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About the Author Ralf G Thiele Ralf G Thiele is currently Associate Professor of Medicine at the University of Rochester (NY, USA). He received a medical degree from Johann-Wolfgang-Goethe University in Frankfurt (Germany). He completed his internship and residency in medicine at Carney Hospital in Boston (MA, USA). He then completed a fellowship in rheumatology at Dartmouth-Hitchcock Medical Center in Lebanon (NH, USA). He has practiced musculoskeletal ultrasound for 20 years and was certified in 1995. He has served as Chair of the Musculoskeletal Section of the American Institute of Ultrasound in Medicine, Chair of the Ultrasound Task Force of the American College of Rheumatology; and has developed and chaired educational ultrasound courses at the University of Rochester, Cooper University Hospital, the American Institute of Ultrasound in Medicine and the American College of Rheumatology. He has developed imaging algorithms, practice guidelines and certification in musculoskeletal ultrasound with the American Institute of Ultrasound in Medicine, the Society of Radiologists in Ultrasound and the American College of Rheumatology.

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Chapter

7 Imaging of gout

Conventional radiography 98

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Computed tomography 100 Dual-energy CT

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MRI

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Imaging of gout provides the practitioner with information beyond physical examination and joint aspiration alone. Typical imaging features of gout are helpful for diagnosis and differential diagnosis and provide information about monosodium urate (MSU) tophus burden, inflammatory activity and associated structural tissue damage. Follow-up imaging of tophi can document changes in volume objectively in response to urate-lowering drugs. Conventional radiographs, ubiquitively available, show characteristic bony defects. Computerized tomography, as a cross-sectional modality, is particularly well suited to detect bony erosions. Color coding of tissues in dualenergy computed tomography scanning helps with the differential diagnosis of crystal arthritis. MRI is best suited to show bony structures and soft tissues at the same time, and contrast enhancement can demonstrate inflammatory changes. Ultrasonography has high resolution and can document tophaceous material, adjacent soft tissues and bony erosions. Doppler sonography can show associated hyperemia.

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Thiele Conventional radiography Conventional radiographs, or x-rays, as they were called by Konrad Roentgen, have been the main diagnostic imaging modality for the detection of gout for over a century. Findings are quite characteristic and can, together with joint aspiration and polarizing microscopy, assist in the diagnosis of gout. Radiographic features of gout include bony erosions with well-defined margins and delicate bony overhangs that may have an ‘egg shell’ appearance (Figure  7.1) [1]. Both features help distinguish these erosions from rheumatoid arthritis, where erosions frequently have irregular, ragged-appearing margins and no new bone formation. Gouty erosions can be seen in typical locations in an asymmetric distribution. Small finger joints, first metatarsal heads and sesamoid bones are often affected, but erosions can affect any other bone area as well. In radiography, 3D structures are projected on 2D films. All bony tissues are superimposed on each other. For detection of erosions, breaks in the bony cortex need to be visualized in profile, or ‘en face’. Overlying bony tissue can obscure cortical lesions if they are rotated out of the radiographic contour. Therefore, different radiographic views are generally obtained. Radiographic findings of gout are quite specific but lack sensitivity. Similar to findings in rheumatoid arthritis, only a small proportion of gouty erosions are detected radiographically compared with cross-sectional imaging [2]. Ultrasonography finds more erosions in gout than conventional radiography, if appropriate high-frequency transducers are used [3]. Moreover, tophaceous deposits are radiolucent and cannot be detected well radiographically. Tophi Figure 7.1. Conventional radiograph with typical bony erosion of gout.

A bony erosion with smooth borders and delicate marginal bony overhangs is seen at the medial aspect of the head of the first metatarsal bone.

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Imaging of gout Figure 7.2. Conventional radiograph with ‘fluffy opacities’ in chronic ­tophaceous gout.

‘Fluffy opacities’ are seen adjacent to the proximal interphalangeal joint in a patient with chronic tophaceous gout.

can sometimes appear as ‘faint, fluffy opacities’ (Figure 7.2) [4]. Over time, tophi can occasionally calcify, and such deposits are then seen on x-rays. Radiography can be helpful in distinguishing gout from chondrocalcinosis, which is associated with calcium-containing deposits. Chondrocalcinosis involving hyaline cartilage can be seen as an irregular band that parallels the bony cortex. This can be seen particularly in the humeral head and over femoral condyles. Radiography can also detect chondrocalcinosis in Figure 7.3. CT scan of forefoot with bony erosion of gout. A

B

(A) Unenhanced computed tomography: axial view of forefoot. Small bony defect is seen over dorsal aspect of first metatarsal head. Tissue with a density between bone and muscle is seen adjacent to bone. (B) Unenhanced computed tomography, same patient. ‘Bone window’ shows in greater detail the erosion of the metatarsal head with overhanging bony margins. 

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Thiele Figure 7.4. 3D rendering of computed tomography of patient with chronic tophaceous gout.

fibrocartilage, particularly the menisci of knees, glenoid labra of shoulder joints, meniscus homologues of the triangular fibrocartilage complex in wrists and acetabular labra of hip joints. Radiation exposure, although low, may limit the number of follow-up studies. The risk of missing the diagnosis needs to be weighed up against the low cost and availability, and can be part of the discussion with the patient. Despite its low sensitivity, there is a role for conventional radiography as a screening modality in suspected gout. Findings of radiography can help distinguish gout from other arthropathies such as rheumatoid arthritis or osteoarthritis. Its advantages include low cost, ubiquitous availability and relatively low radiation exposure.

Computed tomography Computed tomography, or CT scanning, produces cross-sectional images using an x-ray source that rotates around the joint area in question, with a detector on the opposite side. A numerical value (Hounsfield number) can be assigned to the x-ray attenuation [5]. This allows for qualitative and quantitative assessment of soft tissues (Figures 7.3A & 7.3B). Although CT scanning uses x-ray technology, and urate tophi are not usually visible on conventional radiographs, the ability to quantify attenuation allows for characterization of tophaceous material. If software for 3D rendering is available, tophus size and volume may be documented (Figure 7.4). However, tophi may contain calcifications and the distinction of tophi from other calcified lesions may be difficult. CT is useful in differentiating tophi from other soft-tissue masses such as xanthomas or rheumatoid nodules, which have a lower attenuation than tophi [6]. CT scanning overcomes the inherent disadvantages of conventional radiographs, as bony tissues are not superimposed on each other. This allows for a more sensitive, detailed depiction of typical erosions of gout. Punched-out lesions with marginal overhangs, as characterized by Martel [1], are seen in a near 2D plane. This can assist with a diagnosis of gout if other means are inconclusive. 3D rendering of CT images can potentially demonstrate MSU tophi. If this allows for the assessment of tophus volume, it could help estimate the urate

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Imaging of gout Figure 7.5. MRI scan of ankle with tophus and associated synovitis. A

B

  (A) T1 weighted MRI. Chronic tophaceous gout. Tophus is indistinguishable from surrounding synovitis (arrow). (B) T2 fat-suppressed MRI. Tophus has properties different from surrounding tissues (arrow).

burden. Observing the volume of tophi over time would provide an objective measure of treatment response. Cost and potentially radiation exposure may limit widespread use of conventional CT scanning as a measure of treatment response. Furthermore, the sensitivity of 3D CT rendering to detect and observe MSU tophi is not well established. Early studies indicate a decreased sensitivity of three-dimensionally rendered CT compared with other imaging modalities [7]. When urate deposits are found on the surfaces of articular cartilage with arthroscopy, these can be difficult to identify on MRI and CT images, even retrospectively [8]. Imaging that exposes the patient to ionizing radiation should be used judiciously. At 2007 rates, 1.5–2% of all cancers in the USA could be attributed to the use of CT [9]. For typical imaging of gout, irradiation doses vary considerably for different body areas. For assessment of tophi and erosions, the average effective dose is 6 mSv for a spinal CT, 6 mSv for pelvic CT and 0.1 mSv for extremity CT imaging [10].

Dual-energy CT In dual-energy CT (DECT) scanning, two x-ray tubes and two corresponding detectors are used. Two datasets are acquired concurrently. This minimizes errors caused by misregistration or patient movement. Scanning protocols that allow color coding of the composition of tissues are available. This allows distinction of urate tophi and calcified tissue, including bone. Color coding and dual datasets allow a computerized 3D reconstruction of tophaceous deposits with assessment of volumes [11]. This may be an

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Thiele Figure 7.6. T2 fat-suppressed MRI.

advantage over the more operatordependent volume assessment with MRI, in which consecutive magnetic resonance images are traced manually [12]. DECT can distinguish tophaceous material from tendon fibers and has been used to detect MSU tophi in tendons around the ankle [13]. A study that compared quality and radiation exposure of DECT and conventional singleenergy CT for the assessment of lower extremity tendons found DECT to significantly escalate patient exposure to ionizing radiation and provide inferior signalto-noise and contrast-to-noise ratios, irrespective of the different protocols that were assessed [14]. Typical radiation doses for DECT assessment of tendons in foot and ankle of 9.2 mGy (mSv), and for hand and Tophus and surrounding halo seen proximal to wrist of 9.1 mGy (mSv) have been reported calcaneus (arrow). by the manufacturer Siemens [15]. However, lower doses comparable with those of conventional CT scanning have been reported by Choi et al. [16].

MRI MRI can visualize soft tissues and bony structures. MSU tophi and their relationship to surrounding soft-tissue structures can be appreciated. The MRI appearance of tophaceous gout is nonspecific [17–19]. The signal intensity of a tophus will be nearly isointense to that of muscle on T1-weighted images. Findings on T2-weighted images are more variable and can range from homogeneous high-intensity signal to nearhomogeneous low-intensity signal, with an intermediate-to-low heterogeneous signal intensity pattern on T2-weighted images found most commonly (Figures  7.5A,  7.5B &  7.6) [15] . MRI is sensitive in detecting tophi, but the MRI appearance of tophi can be similar to that of other soft-tissue masses, such as giant-cell tumors, which decreases its specificity. High-frequency ultrasound cannot penetrate the bony cortex, but MRI may assess bone marrow edema in the vicinity of MSU tophi and can help understand the pathogenesis of erosions in gout. Inflamed tissues associated with MSU deposits can be appreciated using MRI [20]. The sensitivity of MRI to change of tophi over time is not established.

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Imaging of gout Figure 7.7. Ultrasound. Lateral long axis view of lateral femoral condyle.

Tophus and bony erosion with marginal overhangs. Fluid collection in adjacent recess of knee joint.

Ultrasonography In ultrasonography, sound waves are sent into tissues and reflected at interfaces within and between tissues. The reflected sound waves are detected by the transducer and transformed into pixels of varying brightness on the screen, depending on the strength of the detected signal. Aggregates of calciumcontaining crystals and MSU crystals strongly reflect sound waves. Both are easily visible sonographically. In addition, tissues and vasculature adjacent to crystal aggregates can be visualized in considerable detail [21]. Power Doppler and color Doppler ultrasound can assess hyperemia associated with gout. Ultrasound is sensitive for the detection of bony erosions and can provide information about the tissues and vascularity associated with erosion formation (Figures 7.7 & 7.8). This can help with our understanding of the pathophysiology of erosive gout [22]. Of the available imaging modalities, high-frequency ultrasonography has the highest two-point discrimination (resolution). UltraSonographic appearance of tophaceous sonographic features of gout include detecmaterial: hypoechoic to hyperechoic tion of hypo- to hyper-echoic MSU tophi. inhomogeneous material often surrounded by a small Typical locations that are readily accessible anechoic rim. Tophi often have a characteristic sonographically include the dorsal and sonographic appearance of ‘wet sugar clumps’. medial recess of the first metatarsophalanErosions: breaks in the hyperechoic outline of the geal joints, DIP, PIP and MCP joints of the bony cortex, seen in two perpendicular planes.

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Thiele Figure 7.8. Ultrasound. Same patient and location as in Figure 7.7.

Doppler studies show hyperemic tissue adjacent to monosodium urate tophus.

hands, the fourth extensor compartment of the dorsum of the wrist, insertion of quadriceps tendon, prepatellar bursa, origin and insertion of patellar tendon and insertion of Achilles’ tendon. If hypersaturation of synovial fluid leads to precipitation of MSU crystals within the joint cavity, the hyaline Figure 7.9. Ultrasound. Plantar long axis view of first metatarsophalangeal joint.

Double contour sign in patient with chronic tophaceous gout.

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Imaging of gout Double contour sign: a hyperechoic, irregular cartilage provides a surface for MSU crystal band over the superficial margin of the deposition. This phenomenon leads to a articular cartilage of metatarsal heads, metacarpal sonographic ‘double contour’ of hyperechoic heads, femoral condyles and humeral head. The bony bony cortex, overlying anechoic hyaline carcontour appears hyperechoic (bright), covered by tilage and a parallel hyperechoic layer of MSU anechoic (dark)-appearing hyaline cartilage. This in turn is covered by a hyperechoic (bright)-appearing crystals (Figure 7.9) [21]. This sonographic sign layer of crystals. is sensitive to change. If serum urate concentrations can be maintained below the saturaDoppler ultrasonography assesses the tion point, dissolution of MSU crystal aggrefrequency shift of sound waves reflected off gates can be documented sonographically [23]. moving objects as they pass under the ultrasound Bony erosions can be seen sonographically transducer (Doppler effect). These objects are as breaks in the hyperechoic bony cortex, and primarily erythrocytes. Doppler sonography can should be documented in two perpendicular assess hyperemia in inflamed tissues. Power Doppler ultrasound encodes the strength (power) of blood planes [24]. Ultrasonography detects more flow, but not the direction. erosions in gout than conventional radiography [25,26]. Transducer frequencies of 12 MHz and higher are recommended for the ultrasound assessment of musculoskeletal conditions in general, and for the assessment of gout in particular. In a study that used a gray-scale transducer frequency of 7.5 MHz, ultrasound was not sensitive for typical changes of gout [2]. Inter-operator reliability of ultrasound assessment of gout was found to be excellent [27].

Table 7.1. Sonographic terminology. Term

Definition

Echogenicity

Degree of reflection of sound waves. Brightness of tissues on ultrasound screen Increased reflectivity of tissues leads to increased signal reception, depicted on screen as brightness of pixels

Hypoechoic

Lesser reflection of sound waves than average (or fatty tissue). Darker appearance on screen

Water-containing tissue (e.g., edematous tissue or proliferative synovial tissue)

Hyperechoic

More reflection of sound waves than average. Brighter appearance on screen

Calcium-containing tissue (e.g., bony cortex). Crystalline material including calcium-containing and monosodium urate crystal aggregates

Anechoic

No reflection of sound waves. Dark, or black appearance on screen

Fluid with low cell content (e.g., synovial fluid)

Color or power Doppler ultrasound

Shift of frequency of sound waves reflected from moving object passing the observer (erythrocytes passing under transducer)

Blood flow in vasculature can be color coded for direction. Strength (power) is displayed monochromatically. Color or power Doppler ultrasound characterizes inflammation

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Thiele Financial & competing interests disclosure RG Thiele has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Summary. ƒƒ Conventional radiography is ubiquitously available and shows bony erosions with typical overhanging margins. Sensitivity is limited. ƒƒ CT scanning provides detailed resolution of bony structures. It can be used as the reference modality for the detection of bony erosions. Assessment of inflammatory changes is limited. ƒƒ Color coding of tissues via use of dual-energy CT scanning allows visualization of monosodium urate tophi. Protocols and radiation exposure have not been standardized yet. ƒƒ MRI can visualize tophi, associated inflammatory changes, bony defects and bone marrow changes at the same time. Findings in gout are often nonspecific. ƒƒ Ultrasonography can show monosodium urate tophi, adjacent tissues and bony erosions. Power Doppler ultrasound can show hyperemia of tophus-associated tissues. Ultrasonography has the highest resolution of the available imaging modalities.

References 1

Martel W. The overhanging margin of bone: a roentgenologic manifestation of gout. Radiology 91(4), 755–756 (1968).

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Carter JD, Kedar RP, Anderson SR et al. An analysis of MRI and ultrasound imaging in patients with gout who have normal plain radiographs. Rheumatology 48(11), 1442–1446 (2009).

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Thiele RG, Schlesinger N. Ultrasound detects more erosions in gout than conventional radiography. Arthritis Rheum. 62(Suppl. 10), S368–S369 (2010). Gerster JC, Landry M, Duvoisin B, Rappoport G.

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Computed tomography of the knee joint as an indicator of intraarticular tophi in gout. Arthritis Rheum. 39(8), 1406–1409 (1996).

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Chen CK, Yeh LR, Pan HB et al. Intra-articular gouty tophi of the knee: CT and MR imaging in 12 patients. Skeletal Radiol. 28(2), 75–80 (1999).

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Atlas SW. Exposure to ionizing radiation and estimate of secondary cancers in the era of high-speed CT scanning: projections from the Medicare population. J. Am. Coll. Radiol. 9(4), 245–250 (2012).

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Eftekhari A et al. Dual energy computed tomography in

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About the Authors Puja P Khanna Puja P Khanna is Assistant Clinical Professor of Rheumatology at the University of Michigan (USA). She is dually appointed at the Veterans Affairs Medical Center (MI, USA). She graduated from Sechenov Moscow Medical Academy in Moscow, Russia and completed her residency in medicine at Wright State University (OH, USA), followed by a fellowship in rheumatology at the University of California in Los Angeles (USA). Her research is funded by the American College of Rheumatology (ACR) and focuses on outcomes in arthritides. Her goals are to design patient and physician interventions that improve patient-reported outcomes in gout patients. She is one of the lead authors on the ACR-commissioned guidelines for the management of gout. She also serves as a principal investigator for gout clinical trials. In addition, she is well published in peer-reviewed professional journals, and has written reviews, book chapters and abstracts.

Dinesh Khanna Dinesh Khanna is tenured Associate Professor of Medicine and Director of the University of Michigan Scleroderma Program. He has published over 150 peer-reviewed articles and book chapters. He is a health services researcher with current research focus on developing, validating and refining outcome measures in rheumatic diseases and designing controlled trials. His areas of interest include scleroderma and gout. He is leading the development of patient-reported outcome measures in rheumatic diseases and developing composite response index for clinical trials in scleroderma; both projects are funded by the US NIH. He is also the lead author on the ACR-commissioned guidelines for management of gout.

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Chapter

8 Outcome measures in gout

Acute gout

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Chronic gout

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Other measures

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Puja P Khanna & Dinesh Khanna Gout is known to have a wide spectrum of presentation that ranges from infrequent acute painful attacks to chronic persistent swelling and pain, and once it becomes tophaceous due to deposition of urate crystals in the joints and soft tissues, can result in disability. The immediate goal of therapy is to ameliorate the acute arthritis, and is typically accomplished with NSAIDs, steroids and/or colchicine. The long-term therapy can be complex, where it is necessary to prevent recurrent gout flares and the resulting joint destruction by lowering serum urate. This can be accomplished through lifestyle modifications as well as pharmacologic therapy.

doi:10.2217/EBO.12.234

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Khanna & Khanna An outcome measure is defined in this chapter as specific key measurements (e.g.,  laboratory tests, patient-reported outcomes [PROs] and imaging) used to measure the effect of experimental treatment in a study. An outcome measure should be feasible, reliable and valid [1–3]. A feasible measure is accessible, easily interpretable and associated with low cost. Reliability (precision) is the extent to which a measure yields the same score each time it is administered, if the underlying health condition has not changed. Reliability can be assessed by conducting test– retest (where scores are assessed at two different time points), and assessing internal consistency (assesses correlation between items within each scale). Validity is the extent to which the score a health measure yields accurately reflects the health concept and includes face (sensible), content (comprehensive), construct (measures or correlates with a theorized health construct) and criterion (predicts or correlates with gold standard) validity. Sensitivity to change (also known as responsiveness to change) assesses if an instrument score changes in the right direction when underlying health construct changes; the ability of an instrument to detect clinically important change is crucial to their usefulness as an outcome measure in a clinical trial.

Outcome measures: specific key measurements (e.g., laboratory tests, patient-reported outcomes and imaging) used to measure the effect of experimental treatment in a study.

Table  8.1 presents the instruments that are available for randomized controlled trials (RCTs) and clinical care in patients with gout. Most of these instruments have been included in different RCTs of acute and chronic gout and endorsed by the Outcome Measures in Rheumatology (OMERACT) [4–10].

Acute gout Acute gout trials have largely included patients with an acute onset of gout (self-reported and/or physician-confirmed). These patients have been recruited within 48 h (~50%) to 5 days (~20%) after onset of gout attack as shown in a systematic review [11]. There are no data on reliability (test– retest) for measurements used in acute gout trials; however, this concept may not be important in acute gout due to dynamic changes in signs and symptoms (based on natural history and treatment). Pain Pain is a cardinal symptom of acute gout that is used as the primary outcome in all RCTs for acute gout. It has face and content validity in acute gout. Pain has been captured either as a Likert scale (0 = none; 1 = mild; 2 = moderate; 3  =  severe; and 4  =  extreme), 11-point numeric rating scale (0–10) or visual analog Feasible, reliable and valid outcome measures scale ranging from zero (no pain) to 100 have been developed for clinical trials in gout.

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Outcome measures in gout Table 8.1. Proposed outcome measures for clinical trials for acute and chronic gout. Instrument

Ready for RCTs?

Easily captured in clinical practice?

Yes‡

No

Yes

‡

Yes

Yes

‡

No

HRQoL SF-36 health survey† Health Assessment Questionnaire – Disability Index GAQ 2.0

†

†

PROMIS item banks

Yes

Yes

Patient global assessment using VAS, NRS or Likert scale

Yes§‡

Yes

Investigator global assessment using VAS, NRS or Likert scale¶

Yes§‡

Yes

Patient global assessment of response to treatment

Yes§‡

Yes

Investigator global assessment of response to treatment¶

§‡

Yes

Yes

Patient assessment of pain using VAS, NRS or Likert scale

Yes§‡

Yes

Patient-reported number of acute flare

Yes§‡

Yes

Yes‡

Yes

Yes§‡

Yes

Yes

Yes

Global assessment

Laboratory test Serum urate