Biliary microlithiasis, sludge, crystals, microcrystallization, and usefulness of assessment of nucleation time

Ragama, Sri Lanka

Biliary microlithiasis, sludge, crystals, 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.

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.

CONCLUSIONS: Biliary crystals are associated with gallstone disease, idiopathic pancreatitis, sphincter of Oddi dysfunction, unexplained biliary pain, and post-cholecystectomy biliary pain. Pathways of cholesterol super-saturation, crystallisation, and gallstone formation have been described with scientific 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)

biliary microlithiasis; sludge; crystals; gallstones; idiopathic pancreatitis; sphincter of Oddi dysfunction

Introduction

The 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 as clacium microspheroliths.[1]These are known to have an association with the formation of gallstones, sphincter of Oddi dysfunction, unexplained biliary pain, 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 is increasingly important in the understanding of the cholesterol nucleation process in cholesterol supersaturation, leading to cholesterol crystallization and ultimately the formation of microlithiasis.[9-12]This article aimed to systematically review the available literature on biliary microlithiasis, sludge, and crystals as well as the significance of biliary microscopy and nucleation time and their clinical relevance.

Data sources

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 it was impossible to categorise the data into tables or flow charts.

Biliary microlithiasis, microcrystals, and biliary sludge

The term biliary microlithiasis denotes the presence of small stones in the gallbladder or biliary tree that is ≤3 mm and is not imaged on conventional ultrasonography or cholecystography.[4,5,10]The presence of biliary crystals is not equivalent to microlithiasis but suggests that the bile is supersaturated with cholesterol and that it has nucleated. Thus, the assumption is that if there are biliary crystals on microscopic examination, then there might be microliths.[10-12]Biliary microlithiasis 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]

Detection, etiology, and clinical significance of biliary sludge

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 is associated with pregnancy, with rapid weight loss, particularly in the obese, and with critical illness involving low or absent oral intake, use of total parenteral nutrition, or after gastric surgery. It is also associated with biliary stones with common bile duct obstruction, with certain drugs such as ceftriaxone and octreotide, and with bone marrow or solid organ transplantation.

The clinical course of biliary sludge varies. It often disappears, particularly if the cause is removed. Other cases wax and wane and some may eventually develop gallstones. Complications caused by biliary sludgeinclude 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 apositive 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]

Clinical significance of microcrystals

Gallstone disease is a major health burden in the western world with about 10% of the population affected, mainly by cholesterol stones.[5,17,18]According to the literature, biliary crystals are present in 95% of patients with symptomatic gallstone disease.[5,16-18]It has also been found that about 8% of individuals without gallstones harbor microliths.[11]It has been suggested that the detection of cholesterol monohydrate crystals or calcium bilirubinate granules in bile indicates either the presence of occult gallstones or biliary microlithiasis.[11-13]Biliary microcrystals are present in the gallbladder or duodenal bile in patients with detectable gallbladder calculi, in those with biliary sludge, and in a subset of patients without gallstones.[14-16]

It is also believed that in 25% to 60% of patients, biliary and pancreatic sphincter function might be altered by mechanical irritation by microcrystals and subsequent inflammation, which may enhance gallstone formation.[2,11,16,20]Also, it is believed that microcrystals are a chief contributory cause of unexplained biliary pain in post-cholecystectomy patients. Smaller gallstones carry a higher risk of pancreatitis than larger ones and this supports the possible role of biliary microlithiasis in its etiology. The data reveal that between 7% and 79% of patients with "idiopathic pancreatitis" harbor biliary microlithiasis.[2,11,16,20]

Discussion

The review revealed that biliary microcrystals are a causative factor for gallstone disease. Furthermore, there 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, most studies agree that cholesterol crystallization pathways are dependent on salt/lecithin ratio, bile salt hydrophobicity, temperature, and total lipid concentration. The main drawback is that most of these studies are undertaken in synthesized bile whereas studies of human bile are rare. Overall, all of the data agree that cholesterol crystallization is the initial step in gallstone formation.[5-7,27,28]Even though the pathways of cholesterol crystallization are complex, the formation of gallstones from crystallized bile is well correlated and the contributory factors of cholesterol supersaturation and crystallization for stone formation have been agreed upon.[6, 27, 37-39]

Since the majority of the data revealed that biliary microcrystals are associated with gallstone formation, idiopathic pancreatitis, post-cholecystectomy biliary pain, and sphincter of Oddi dysfunction, microscopic detection seems to be beneficial.[32,40-42]The review shows that biliary microscopy for microcrystals is sensitive and specific for risk stratification of microcrystal-associated disease conditions. Studies have shown that microscopic examination of bile is more sensitive and specific than oral cholecystography or trans-abdominal ultrasonography in detection of biliary sludge in patients with idiopathic pancreatitis, unexplained biliary pain, and acalculous gallbladder disease.[4,30,32,34,40]

Universally there is no controversy over the definition of nucleation time of bile. The value of bile nucleation time in predicting the risk of cholesterol crystallization, which eventually leads to gallstone formation, has been well recognized. By definition,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.

Funding: None.

Ethical approval: This study was approved by the Ethics Committee of the Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka.

Contributors: AV wrote the main body of the article under the supervision of DKI. NNMM provided advice on medical aspects. AV is the guarantor.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received February 8, 2010

Accepted after revision April 10, 2010

Author Affiliations: Department of Clinical Anatomy (Abeysuriya V) and Department of Surgery (Deen KI), Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka; Department of Gastroenterology, National Hospital of Sri Lanka, Colombo, Sri Lanka (Navarathne NMM)

Dr. Vasitha Abeysuriya, MBBS, Lecturer/Surgical Registrar, Department of Clinical Anatomy, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka (Tel: 94-1-77-3528167; Email: vasitha76@ hotmail.com)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.