This
systematic review aimed to highlight the importance of biliary
microlithiasis, sludge, and crystals, and their association with
gallstones, unexplained biliary pain, idiopathic pancreatitis,
and sphincter of Oddi dysfunction.
This
systematic review aimed to highlight the importance of biliary
microlithiasis, sludge, and crystals, and their association with
gallstones, unexplained biliary pain, idiopathic pancreatitis,
and sphincter of Oddi dysfunction.
This
systematic review aimed to highlight the importance of biliary
microlithiasis, sludge, and crystals, and their association with
gallstones, unexplained biliary pain, idiopathic pancreatitis,
and sphincter of Oddi dysfunction.
microcrystallization, and usefulness of assessment of nucleation time Vasitha Abeysuriya, Kemal I Deen and Navarathne MM Navarathne Ragama, Sri Lanka
BACKGROUND: The process of microcrystallization, its sequel
and the assessment of nucleation time is ignored. This systematic review aimed to highlight the importance of biliary microlithiasis, sludge, and crystals, and their association with gallstones, unexplained biliary pain, idiopathic pancreatitis, and sphincter of Oddi dysfunction. DATA SOURCES: Three reviewers performed a literature search of the PubMed database. Key words used were "biliary microlithiasis", "biliary sludge", "bile crystals", "cholesterol crystallisation", "bile microscopy", "microcrystal formation of bile", "cholesterol monohydrate crystals", "nucleation time of cholesterol", "gallstone formation", "sphincter of Oddi dysfunction" and "idiopathic pancreatitis". Additional articles were sourced from references within the studies from the PubMed search.
support. Bile microscopy is a useful method to detect
microcrystals and the assessment of nucleation time is a good method of predicting the risk of cholesterol crystallisation. (Hepatobiliary Pancreat Dis Int 2010; 9: 248-253) KEY WORDS: biliary microlithiasis; sludge; crystals; gallstones; idiopathic pancreatitis; sphincter of Oddi dysfunction
Introduction
he clinical value of biliary microscopy and the
related clinical significance of biliary microlithiasis, microcrystals, and biliary sludge have been neglected. Furthermore, it has been observed that the demarcation of the definitions of biliary microlithiasis, microcrystals, and biliary sludge is not clear in most instances. Microlithiasis refers to small gallstones. Biliary sludge is an ultrasonographic diagnosis. Biliary crystals are microscopic structures and include cholesterol monohydrate and calcium bilirubinate crystals, as well CONCLUSIONS: Biliary crystals are associated with gallstone [1] disease, idiopathic pancreatitis, sphincter of Oddi dysfunction, as clacium microspheroliths. These are known to unexplained biliary pain, and post-cholecystectomy biliary have an association with the formation of gallstones, pain. Pathways of cholesterol super-saturation, crystallisation, sphincter of Oddi dysfunction, unexplained biliary pain, and gallstone formation have been described with scientific and idiopathic pancreatitis.[1, 2] Rapid crystallisation of cholesterol in bile is a key factor in cholesterol gallstone formation.[3-8] The nucleation time and method of its assessment Author Affiliations: Department of Clinical Anatomy (Abeysuriya V) is increasingly important in the understanding of and Department of Surgery (Deen KI), Faculty of Medicine, University of the cholesterol nucleation process in cholesterol Kelaniya, Ragama, Sri Lanka; Department of Gastroenterology, National supersaturation, leading to cholesterol crystallization Hospital of Sri Lanka, Colombo, Sri Lanka (Navarathne NMM) and ultimately the formation of microlithiasis.[9-12] This Corresponding Author: Dr. Vasitha Abeysuriya, MBBS, Lecturer/Surgical Registrar, Department of Clinical Anatomy, Faculty of Medicine, University article aimed to systematically review the available of Kelaniya, Ragama, Sri Lanka (Tel: 94-1-77-3528167; Email: vasitha76@ literature on biliary microlithiasis, sludge, and crystals hotmail.com) as well as the significance of biliary microscopy and nucleation time and their clinical relevance. 2010, Hepatobiliary Pancreat Dis Int. All rights reserved. RESULTS: We found that biliary microcrystals account for almost all patients with gallstone disease, 7% to 79% with idiopathic pancreatitis, 83% with unexplained biliary pain, and 25% to 60% with altered biliary and pancreatic sphincter function. Overall, the detection of biliary microcrystals in gallstone disease has a sensitivity ranging from 55% to 87% and a specificity of 100%. In idiopathic pancreatitis, the presence of microcrystals ranges from 47% to 90%. A nucleation time less than 10 days in hepatic bile or ultra-filtered gallbladder bile has a specificity of 100% for cholesterol gallstone disease.
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Biliary microlithiasis, sludge and crystals
Data sources
contributes to gallstone formation in the extra-hepatic
biliary system, idiopathic pancreatitis, and sphincter of Oddi dysfunction.[10, 13-18] Biliary crystals are microscopic structures and include cholesterol monohydrate and Ca bilirubinate crystals, as well as Ca microspheroliths. Cholesterol crystals may be either cholesterol monohydrate or nonhydrate.[1,4,9] Classically, cholesterol monohydrate crystals are plates.[1,4,9] Crystal forms, filamentous, helical, arcs, and tubular are known as non-hydrated cholesterol crystals.[5,17-24] Data indicate that these atypical forms of crystal morphology predominate in some patients with a clinical diagnosis of acalculous gallbladder disease.[24-26] Calcium bilirubinate is a major constituent of pigment stones and consists of the calcium salts of unconjugated bilirubin. Thus, calcium bilirubinate is the other predominant constituent of biliary crystals, and appears as black, brown, or light brown aggregates on light microscopy.[5-7, 24, 27] Biliary sludge is an ultrasonographic diagnosis when there is echogenic material that layers in the gallbladder and does not cast acoustic shadows; also it is a mixture of particulate solids that have precipitated from bile.[5, 16] Such sediment consists of cholesterol crystals, calcium bilirubinate pigment, and other calcium salts.[5, 21] According to the data, biliary sludge is sediment composed of cholesterol monohydrate crystals and bilirubin granules embedded in a matrix of mucus gel.[5, 10, 16] Mucus hypersecretion and early glandular metaplasia in the epithelium are observed in gallbladders harboring sludge, indicating that in patients with sludge, the gallbladder is abnormal, showing mucus hypersecretion and glandular metaplasia.[5, 18, 21]
We performed a systematic review of the current evidence
on biliary microlithiasis, sludge, and crystals, and their association with gallstones, unexplained biliary pain, idiopathic pancreatitis, and sphincter of Oddi dysfunction. Three qualified reviewers searched the PubMed database to identify studies of biliary microlithiasis, bile microscopy, and cholesterol nucleation time from 1983 to 2010. Within PubMed, the MeSH browser was used by entering the key words: bile microscopy, microlithiasis, biliary sludge, microcrystal formation of bile, cholesterol monohydrate crystals, nucleation time of cholesterol, gallstone formation, and idiopathic pancreatitis. Studies were limited to those written in English. Agreement among the investigators was essential for data inclusion and few minor disagreements were resolved. The abstracts of the studies found in the search were analyzed to judge their relevance. Case reports, technical reports, articles with insufficient clinical data, series duplicating previously published data from the same author or institution, and papers that did not contain the data needed for this review were excluded. From the references of the included papers, further relevant articles that were not identified by the PubMed search, were added to the review. Papers reporting the same data were identified and those with the most detailed information were selected. The possibility of combining the results of the studies was explored, but there were several obstacles. Lack of prospective studies and randomization precluded a formal meta-analysis. Moreover, a significant proportion of the studies undertaken in the same institution did not provide sufficient data even to attempt combination, and those that provided such data were statistically heterogeneous. Therefore, the results of the studies are presented separately under different subheadings, since Detection, etiology, and clinical significance of it was impossible to categorise the data into tables or biliary sludge flow charts. Sludge is usually detected by trans-abdominal ultra-sonography.[16, 21] Endoscopic ultrasonography is more sensitive in the detection of biliary sludge.[10, 16, 21] Biliary sludge Biliary microlithiasis, microcrystals, and is associated with pregnancy, with rapid weight loss, biliary sludge particularly in the obese, and with critical illness involving The term biliary microlithiasis denotes the presence of low or absent oral intake, use of total parenteral nutrition, small stones in the gallbladder or biliary tree that is 3 or after gastric surgery. It is also associated with biliary mm and is not imaged on conventional ultrasonography stones with common bile duct obstruction, with certain or cholecystography.[4, 5, 10] The presence of biliary drugs such as ceftriaxone and octreotide, and with bone crystals is not equivalent to microlithiasis but suggests marrow or solid organ transplantation. The clinical course of biliary sludge varies. It often that the bile is supersaturated with cholesterol and that it has nucleated. Thus, the assumption is that if there disappears, particularly if the cause is removed. Other are biliary crystals on microscopic examination, then cases wax and wane and some may eventually develop there might be microliths.[10-12] Biliary microlithiasis gallstones. Complications caused by biliary sludge Hepatobiliary Pancreat Dis IntVol 9No 3 June 152010 www.hbpdint.com 249
Hepatobiliary & Pancreatic Diseases International
include biliary colic, acute cholangitis, and acute
pancreatitis.[10, 16, 18, 22, 23]
Crystallization of cholesterol in bile and
gallstone formation Cholesterol crystallization is the primary step in the pathogenesis of cholesterol gallstone formation. Although crystallization pathways have been studied extensively in model bile, limited data are available on crystallization pathways in human bile.[4, 24, 27] Several pathways of cholesterol crystallization have been described.[4, 17] Some studies have described five crystallization pathways in model biles depending on salt/lecithin ratio, bile salt hydrophobicity, temperature, and total lipid concentration.[4, 27, 28] It has been suggested that two putative pathways of cholesterol nucleation in bile might involve precipitation of classic plate-like cholesterol crystals from multilamellar vesicles and nucleation of anhydrous cholesterol occurring internally as a liquid core within unilamellar vesicles, also known as internal nucleation.[27, 28] Furthermore, recent data indicate that solid cholesterol precipitates as crystal forms other than plate-like structures.[4, 5, 12, 15] The true micromolecular sequence of cholesterol crystallization and stone formation has not been exactly identified. According to studies, the proposed sequence of events is that the cholesterol is carried out in mixed micelles and vesicles.[4, 15, 28] The micelles are aggregates of phospholipid, bile salts, and cholesterol, and the vesicles are closed spherical bilayers of phospholipids with associated cholesterol. In the micelles and vesicles, polar groups such as hydroxyl and phosphate face the aqueous solvent, whereas nonpolar, hydrophobic groups are hidden inside. The vesicles have cholesterolrich areas that can, when they come into contact, form cholesterol crystals. At higher cholesterol concentrations, multilamellar vesicles of cholesterol crystals form on the surfaces of these vesicles and grow within the mucin gel. Cholesterol crystals are glued together by bile proteins to make stones.[4, 5, 15, 17]
Bile nucleation time and assessment
Nucleation time is the terminal step in studies examining bile lithogenicity. The number of days taken for microscopic plate-like cholesterol monohydrate crystals to appear in human bile has been referred to as the crystal nucleation time. Most data agree that if biliary microcrystals appear in less than 10 days, the
bile should be considered potentially lithogenic.[19, 29, 30]
Also, it has been shown that the nucleation time of ultrafiltered bile has a specificity of 100% for cholesterol stones.[11, 28] Also, in those with cholesterol stones, nucleation time has been shown to be shorter in those with multiple cholesterol stones than in those who have a solitary stone.[6, 7, 28] Most clinical studies have utilized duodenal bile for microscopy for the simple reason that it is risky to aspirate bile from the gallbladder and it is unethical to operate on a patient for this purpose. Moreover, surgeons do not prefer to aspirate bile from the gallbladder for fear of a bile leak. The data suggest that duodenal bile is less suitable for determination of nucleation time because of contamination with pancreatic enzymes, which may also contaminate nasobiliary bile. Contamination of bile with pancreatic enzymes is known to alter the nucleation time. Therefore, the advocated approach of collecting bile for assessment of nucleation time is either during bile duct aspiration at surgery or by ERCP if possible.[14, 29, 31] In 20 cases of biliary colic that had a normal oral cholecystogram and ultrasound, 11 (55%) showed microcrystals in the form of cholesterol monohydrate crystals and/or calcium bilirubinate granules on polarized light microscopy of the duodenal bile.[28] Microcrystals were noted in gallbladder bile of all cases of proven gallstones but not in the duodenal bile samples from healthy subjects.[27, 28] Another study, in which gallbladder bile was collected by duodenal intubation or during surgery, compared 16 patients who were free of stones with 17 patients with proven stones, and showed that none of the 16 patients had cholesterol monohydrate crystals or calcium bilirubinate granules compared with 13 of the 17 patients, who had cholesterol monohydrate crystals. Only 2 of 4 patients with pigment stones in the study had calcium bilirubinate granules.[14, 23] Review of the data showed that according to a study of 31 patients with idiopathic pancreatitis, 23 (74%) had biliary sludge and microcrystals consisting of cholesterol monohydrate and calcium bilirubinate microcrystals. Also in the same study, biliary sludge was detected only in 11 (47%) of these patients by trans-abdominal ultrasonography.[26, 28] According to a study of 72 patients to evaluate the usefulness of microscopic examination of pure bile directly collected from the biliary tract during endoscopic retrograde cholangiopancreatogram without hormonal stimulation, the presence of microcrystals exhibited a sensitivity and specificity for cholelithiasis recognition of 82.7% and 100%, respectively, with a
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Biliary microlithiasis, sludge and crystals
positive predictive value of 88%.[31]
According to the literature, the prevalence of biliary microcrystals in patients with idiopathic pancreatitis (75%) and gallstone pancreatitis (90%) is significantly higher than in those with alcoholic pancreatitis (15%).[32] In the identification of the etiology of gallstone pancreatitis, the presence of microcrystals has a sensitivity of 90%, specificity of 85%, positive predictive value of 85.7%, and negative predictive value of 89.4%. The overall accuracy is 87.5%.[32] Thus assessment of bile microcrystals may provide a clue to the etiology of idiopathic pancreatitis. Elaborating further, a study revealed that cholesterol microcrystals help to identify cholesterol gallstones (sensitivity, 87%; specificity, 97%; positive predictive value, 97%), therefore the presence of cholesterol crystals is significantly related to the cholesterol content of gallstones.[33]
is a significant association between biliary microcrystals
and idiopathic pancreatitis.[21-28] Although available data support an association of biliary microcrystalization with post-cholecystectomy biliary pain and sphincter of Oddi dysfunction, the number of studies is small. However, cumulative data indicate that biliary microcrystals are a causative factor for post-cholecystectomy biliary pain and sphincter of Oddi dysfunction.[18, 23, 29-36] Cholesterol crystals are suggested to be able to cause a functional obstruction at the sphincter of Oddi by inducing papillitis, papillary spasm, or papillary stenosis.[23, 34, 35] Disagreement in reports about the definition of biliary sludge is minimal. According to some studies, the definition of biliary microcrystals is based on both the crystal morphology and morphometry but others consider the definition based on only the crystal morphology, at light and polarized microscopy. [1,4,9,16,17,21,27] There is controversy over the sequence in pathways of cholesterol microcrystal and pigment stone formation. In general, Clinical significance of microcrystals most studies agree that cholesterol crystallization Gallstone disease is a major health burden in the western world with about 10% of the population affected, mainly pathways are dependent on salt/lecithin ratio, bile by cholesterol stones.[5, 17, 18] According to the literature, salt hydrophobicity, temperature, and total lipid biliary crystals are present in 95% of patients with concentration. The main drawback is that most of these symptomatic gallstone disease.[5, 16-18] It has also been studies are undertaken in synthesized bile whereas found that about 8% of individuals without gallstones studies of human bile are rare. Overall, all of the data crystallization is the initial step in harbor microliths.[11] It has been suggested that the agree that cholesterol[5-7, gallstone formation. 27, 28] Even though the pathways detection of cholesterol monohydrate crystals or calcium of cholesterol crystallization are complex, the formation bilirubinate granules in bile indicates either the presence of gallstones from crystallized bile is well correlated and of occult gallstones or biliary microlithiasis.[11-13] Biliary the contributory factors of cholesterol supersaturation microcrystals are present in the gallbladder or duodenal and crystallization for stone formation have been agreed bile in patients with detectable gallbladder calculi, in upon.[6, 27, 37-39] those with biliary sludge, and in a subset of patients Since the majority of the data revealed that biliary without gallstones.[14-16] microcrystals are associated with gallstone formation, It is also believed that in 25% to 60% of patients, idiopathic pancreatitis, post-cholecystectomy biliary biliary and pancreatic sphincter function might be pain, and sphincter of Oddi dysfunction, microscopic altered by mechanical irritation by microcrystals and detection seems to be beneficial.[32, 40-42] The review subsequent inflammation, which may enhance gallstone shows that biliary microscopy for microcrystals formation.[2, 11, 16, 20] Also, it is believed that microcrystals is sensitive and specific for risk stratification of are a chief contributory cause of unexplained biliary microcrystal-associated disease conditions. Studies pain in post-cholecystectomy patients. Smaller gallstones have shown that microscopic examination of bile is carry a higher risk of pancreatitis than larger ones and more sensitive and specific than oral cholecystography this supports the possible role of biliary microlithiasis in or trans-abdominal ultrasonography in detection of its etiology. The data reveal that between 7% and 79% biliary sludge in patients with idiopathic pancreatitis, of patients with "idiopathic pancreatitis" harbor biliary unexplained biliary pain, and acalculous gallbladder microlithiasis.[2, 11, 16, 20] disease.[4, 30, 32, 34, 40] Universally there is no controversy over the definition of nucleation time of bile. The value of bile Discussion nucleation time in predicting the risk of cholesterol The review revealed that biliary microcrystals are a crystallization, which eventually leads to gallstone causative factor for gallstone disease. Furthermore, there formation, has been well recognized. By definition, Hepatobiliary Pancreat Dis IntVol 9No 3 June 152010 www.hbpdint.com 251
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phospholipids and bile acids on cholesterol nucleation time
and vesicular/micellar cholesterol in gallbladder bile of patients with cholesterol stones. J Lipid Res 1993;34:1457-1464. 4 Portincasa P, van Erpecum KJ, Vanberge-Henegouwen GP. Cholesterol crystallisation in bile. Gut 1997;41:138-141. 5 Johnston DE, Kaplan MM. Pathogenesis and treatment of gallstones. N Engl J Med 1993;328:412-421. 6 Ostrow JD. Bilirubin solubility and the etiology of pigment gallstones. Prog Clin Biol Res 1984;152:53-69. 7 Nakai K, Tazuma S, Nishioka T, Chayama K. Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation. Biochim Biophys Acta 2003;1632:48-54. 8 Gustafsson U, Sahlin S, Einarsson C. Biliary lipid composition in patients with cholesterol and pigment gallstones and gallstone-free subjects: deoxycholic acid does not contribute to formation of cholesterol gallstones. Eur J Clin Invest 2000;30: 1099-1106. 9 Peled Y, Halpern Z, Baruch R, Goldman G, Gilat T. Cholesterol nucleation from its carriers in human bile. Hepatology 1988;8: 914-918. 10 Konikoff FM, Laufer H, Messer G, Gilat T. Monitoring cholesterol crystallization from lithogenic model bile by time-lapse density gradient ultracentrifugation. J Hepatol 1997;26:703-710. 11 Gerke H, Baillie J. Biliary microlithiasis: a neglected cause of recurrent pancreatitis and biliary colic? J Gastroenterol Hepatol 2005;20:499-501. 12 S ahoo SK, Tudu D. Role of duodenal bile crystal analysis in idiopathic pancreatitis. Trop Gastroenterol 2001;22:205-206. 13 Ros E, Navarro S, Bru C, Garcia-Pugs A, Valderrama R. Occult microlithiasis in 'idiopathic' acute pancreatitis: prevention of relapses by cholecystectomy or ursodeoxycholic acid therapy. Gastroenterology 1991;101:1701-1709. 14 Petroni ML, Jazrawi RP, Ahmed HA, Finch PJ, Dormandy J, Northfield TC. Cholesterol nucleation time measurement in nasobiliary or nasoduodenal bile. Comparison with surgical bile. Scand J Gastroenterol 1993;28:803-808. 15 Tudyka J, Kratzer W, Maier C, Mason R, Wechsler JG. The Funding: None. relation between biliary lipids, nucleation time, and number Ethical approval: This study was approved by the Ethics Committee of gallbladder stones after percutaneous gallbladder puncture. of the Faculty of Medicine, University of Kelaniya, Ragama, Sri Scand J Gastroenterol 1994;29:844-848. Lanka. Contributors: AV wrote the main body of the article under the 16 Lee SP, Nicholls JF, Park HZ. Biliary sludge as a cause of acute pancreatitis. N Engl J Med 1992;326:589-593. supervision of DKI. NNMM provided advice on medical aspects. 17 Sedaghat A, Grundy SM. Cholesterol crystals and the AV is the guarantor. formation of cholesterol gallstones. N Engl J Med 1980;302: Competing interest: No benefits in any form have been received 1274-1277. or will be received from a commercial party related directly or 18 Portincasa P, van de Meeberg P, van Erpecum KJ, Palasciano indirectly to the subject of this article. G, VanBerge-Henegouwen GP. An update on the pathogenesis and treatment of cholesterol gallstones. Scand J Gastroenterol Suppl 1997;223:60-69. References 19 Sharma BC, Agarwal DK, Baijal SS, Negi TS, Choudhuri G, 1 J ngst C, Kullak-Ublick GA, Jngst D. Gallstone disease: Saraswat VA. Effect of endoscopic sphincterotomy on gall Microlithiasis and sludge. Best Pract Res Clin Gastroenterol bladder bile lithogenicity and motility. Gut 1998;42:288-292. 2006;20:1053-1062. 20 P attinson NR, Willis KE. Nucleation of cholesterol crystals 2 Saraswat VA, Sharma BC, Agarwal DK, Kumar R, Negi TS, from native bile and the effect of protein hydrolysis. J Lipid Tandon RK. Biliary microlithiasis in patients with idiopathic Res 1991;32:215-221. acute pancreatitis and unexplained biliary pain: response to 21 Lee SP, Nicholls JF. Nature and composition of biliary sludge. therapy. J Gastroenterol Hepatol 2004;19:1206-1211. Gastroenterology 1986;90:677-686. 3 Jngst D, Lang T, Huber P, Lange V, Paumgartner G. Effect of 22 Williamson RC. Endoscopic sphincterotomy in the early
nucleation time indicates early aggregation of
cholesterol molecules from supersaturated bile into submicroscopic nuclei, a state which is not seen under a light microscope.[28, 30, 34] This crucial step is followed by precipitation, growth, and aggregation of cholesterol crystals, which become visible under a light microscope.[6, 14, 19, 38] We suggest that the term crystal detection time is more appropriate than crystal nucleation time. Biliary microcrystals are best detected by polarised light microscopy, not ordinary light microscopy. It is revealed that polarized light microscopy increases the sensitivity and specificity of microcrystal detection over conventional light microscopy.[28, 31, 34, 37, 43] Also, the bile samples for studies of microcrystalization provide more accurate data if obtained from the bile ducts, either by ERCP cannulation or by direct aspiration at the time of surgery.[28, 37-39, 44] Duodenal bile is known to be associated with longer nucleation times, likely due to contamination with pancreatic and duodenal secretions, and is generally less suitable for studies of microcrystal detection.[14, 29, 39, 40] In conclusion, more data are needed to confirm the association between biliary microcrystallization and clinical conditions such as idiopathic pancreatitis, biliary pain after cholecystectomy, and sphincter of Oddi dysfunction. The overall results demonstrate that biliary microcrystals are an initial step in the formation of gallstones. Polarised light microscopy is a useful method to detect microcrystals and assessment of nucleation time is a good method of predicting the risk of cholesterol crystallization.
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23 24
25
26 27
28
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31
32
33
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Q uallich LG, Stern MA, Rich M, Chey WD, Barnett JL, Elta GH. Bile duct crystals do not contribute to sphincter of Oddi dysfunction. Gastrointest Endosc 2002;55:163-166. T anaka J, Ise H, Takahashi W, Suzuki N, Sato T, Komatsu H. Crystal morphology of cholesterol monohydrate in cholesterol gallstones and gallbladder bile. Nippon Geka Gakkai Zasshi 1988;89:1834-1842. G ollish SH, Burnstein MJ, Ilson RG, Petrunka CN, Strasberg SM. Nucleation of cholesterol monohydrate crystals from hepatic and gall-bladder bile of patients with cholesterol gall stones. Gut 1983;24:836-844. L andi K, Sinard J, Crawford JM, Topazian M. Cholesterol crystal morphology in acalculous gallbladder disease. J Clin Gastroenterol 2003;36:364-366. D elchier JC, Benfredj P, Preaux AM, Metreau JM, Dhumeaux D. The usefulness of microscopic bile examination in patients with suspected microlithiasis: a prospective evaluation. Hepatology 1986;6:118-122. G ogna A, Kar P, Acharya NR, Anand VJ, Kapoor R. Polarized light microscopic examination of human bile in the diagnosis of microlithiasis of the gallbladder. Trop Gastroenterol 1989;10:167-172. M arks JW, Broomfield P, Bonorris GG, Schoenfield LJ. Factors affecting the measurement of cholesterol nucleation in human gallbladder and duodenal bile. Gastroenterology 1991;101:214-219. H olan KR, Holzbach RT, Hermann RE, Cooperman AM, Claffey WJ. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979;77:611617. B uscail L, Escourrou J, Delvaux M, Guimbaud R, Nicolet T, Frexinos J, et al. Microscopic examination of bile directly collected during endoscopic cannulation of the papilla. Utility in patients with suspected microlithiasis. Dig Dis Sci 1992;37:116-120. C hebli JM, Ferrari Jnior AP, Silva MR, Borges DR, Atallah AN, das Neves MM. Biliary microcrystals in idiopathic acute pancreatitis: clue for occult underlying biliary etiology. Arq Gastroenterol 2000;37:93-101. M iquel JF, Prado A, Asahi H, Ibnez L, Guzmn S, Cruz F, et al. Occult gallbladder disease or microlithiasis in patients
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Chil 1997;125:869-878. 34 Baeyens E, De Teresa J, Gil Extremera B, Gato R, Agredano G, Rodrigo M. Acute pancreatitis and microcrystals. Importance of the bile collection and biochemical parameters. Rev Esp Enferm Dig 1997;89:843-854. 35 Neoptolemos JP, Davidson BR, Winder AF, Vallance D. Role of duodenal bile crystal analysis in the investigation of 'idiopathic' pancreatitis. Br J Surg 1988;75:450-453. 36 R amond MJ, Dumont M, Belghiti J, Erlinger S. Sensitivity and specificity of microscopic examination of gallbladder bile for gallstone recognition and identification. Gastroenterology 1988;95:1339-1343. 37 v an Erpecum KJ, van Berge Henegouwen GP, Stoelwinder B, Stolk MF, Eggink WF, Govaert WH. Cholesterol and pigment gallstone disease: comparison of the reliability of three bile tests for differentiation between the two stone types. Scand J Gastroenterol 1988;23:948-954. 38 de Bruijn MA, Noordam C, Goldhoorn BG, Tytgat GN, Groen AK. The validity of the cholesterol nucleation assay. Biochim Biophys Acta 1992;1138:41-45. 39 P oupon R, Chrtien Y, Darnis F. Cholesterol crystals, cholesterol saturation of bile and biliary lithiasis. Gastroenterol Clin Biol 1984;8:260-263. 40 Kaw M, Brodmerkel GJ Jr. ERCP, biliary crystal analysis, and sphincter of Oddi manometry in idiopathic recurrent pancreatitis. Gastrointest Endosc 2002;55:157-162. 41 Paumgartner G. Biliary physiology and disease: reflections of a physician-scientist. Hepatology 2010;51:1095-1106. 42 Kaur T, Kaur S. Pathophysiological Conditions in Cholelithiasis Formation in North Indian Population: Spectroscopic, Biophysical, and Biochemical Study. Biol Trace Elem Res 2010 Feb 26. [Epub ahead of print] 43 Ardengh JC, Malheiros CA, Rahal F, Pereira V, Ganc AJ. Microlithiasis of the gallbladder: role of endoscopic ultrasonography in patients with idiopathic acute pancreatitis. Rev Assoc Med Bras 2010;56:27-31. 44 K eizman D, Ish-Shalom M, Konikoff FM. The clinical significance of bile duct sludge: is it different from bile duct stones? Surg Endosc 2007;21:769-773. Received February 8, 2010 Accepted after revision April 10, 2010
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