Identifying small pelvic inclusions through SEM technology.

Tiny calcified structures may be occasionally recovered during excavation of human skeletal remains. Since taphonomic processes may displace these structures from their topographical relation with neighbouring organs or bones, differential diagnoses may pose a major challenge to the archeologist and/or anthropologist. Enteroliths, kidney stones or gallstones, phleboliths, calcified ganglia, or sesamoid bones account for most of such calcified tiny structures. In addition to their pure medical/paleopathological interest, some remains may be related to diet, to chronic haemolytic conditions, and/or to infections or chronic intestinal diseases. We here describe the technical procedures carried out to confirm or refute the identification of a sesamoid bone. The object in question was a small (5 × 3 × 2 mm) calcified structure that appeared over the right coxal bone of an 18th century individual buried in the church Nuestra Señora de La Concepción, in Santa Cruz de Tenerife (Canary Islands). For comparative purposes we also analyzed kidney stones and gallstones from modern individuals. As shown in this study, scanning electron microscope (SEM) analysis is the preferred method to establish a precise differential diagnosis in these cases.

A comparative study of mud-like and coralliform calcium carbonate gallbladder stones.

To gain insight to underlying mechanism of the formation of calcium carbonate (CaCO3 ) gallbladder stones, we did comparative study of stones with mud appearance and those with coralliform appearance. A total of 93 gallbladder stones with mud appearance and 50 stones with coralliform appearance were analyzed. The appearance, color, texture, and the detection of Clonorchis sinensis eggs by microscopic examination were compared between the two groups. Then, the material compositions of stones were analyzed using Fourier Transform Infrared spectroscopy and the spectrogram characteristics were compared. Moreover, microstructure characteristics of the two kinds of stones were observed and compared with Scanning Electron Microscopy. Mud-like gallbladder stones were mainly earthy yellow or brown with brittle or soft texture, while coralliform stones were mainly black with extremely hard texture, the differences between the two groups was significant (p < .05). The analytic results of FTIR spectroscopy showed that 95.7% (89/93) of the mud-like gallbladder stones were CaCO3 stones, and mainly aragonite; while all of the coralliform stones were CaCO3 stones, and mainly calcite (p < .05). Meanwhile, microscopic examination indicated that the detection rate of Clonorchis sinensis eggs in mud-like CaCO3 stones was lower than that in coralliform CaCO3 stones (p < .05), and that in aragonite CaCO3 stones was lower than that in calcite CaCO3 stones(p < .05). Mud-like CaCO3 stones mainly happened to patients with cystic duct obstruction. Clonorchis sinensis infection was mainly associated with coralliform (calcite) CaCO3 stones. Cystic duct obstruction was mainly associated with mud-like (aragonite) CaCO3 stones.

Combined microstructural and mineralogical phase characterization of gallstones in a patient-based study in SW Spain - Implications for environmental contamination in their formation.

This study explores the environmental impact of metal exposure on humans through detailed phase and structural characterization of gallstones from two environmentally contrasting populations in Huelva Province (SW Spain). A total of 42 gallstone samples, obtained after surgical intervention at the Riotinto Hospital, were studied by powder X-ray diffraction (XRD), Fourier Transformed Infra-Red spectroscopy (FTIR), FTIR-µ-ATR (Attenuated Total Reflection) coupled with an optical microscope, and by Environmental Scanning Electron Microscope with Energy Dispersive X-ray Spectroscopy (ESEM-EDS), and subsequently classified according to their phase composition and structure. Additionally, the patients were enquired for their living habits in order to analyze the source of possible exposure to metal contamination. The gallstones were classified into pure, mixed and composite cholesterol stones, black and brown pigment stones, and carbonate stones. The patients from the study group residing in a region with acknowledged metal contamination of both natural and anthropogenic origin have a higher risk of metal exposure through contaminated soil, particle matter in the air, and consumption of local water and food products. According to our findings, the metal exposure is related to a higher tendency of forming black pigment stones in the study group in comparison to the control group residing in a natural park with nearly pristine environmental conditions. Moreover, the gallstones from the study group showed to contain more abundant metal components, such as Cu, Fe, Ni, and Zn, than those from the control group. To our knowledge this is the first study to examine the regional environmental impact of metal exposure on human gallstones.

Analysis of gallstone composition and structure in Jharkhand region.

AIM: The aim of this study was to analyze gallstones structurally and chemically as this may help to direct the measures for its treatment. METHODOLOGY: On the basis of morphology, 459 gallstones were categorized into pigmented, cholesterol, and mixed gallstones and analyzed for their chemical structure and composition. Elementary analysis was done with the help of inductively coupled plasma, optical emission spectrophotometry. Fourier transform infrared spectroscopy was used for compound analysis. The effects of thermal stability were analyzed by thermal gravimetric analysis (TGA). Statistical analysis was done to correlate gallstone composition with their shape and number in gallbladder. RESULTS: Pigmented gallstones were predominant and generally occurred in multiple forms, cholesterol as solitaire and black pigmented as slug (χ (2) = 3.56; p < 0.001). Scanning electron microscopy showed the crystals of cholesterol in cholesterol gallstones while pigmented gallstones were more compact in structure. Both types of structure were seen in mixed stones while black pigmented stones were amorphous in nature. This difference in structures might be due to difference in chemical compositions. Cholesterol and mixed stones contained basically cholesterol, brown pigmented constituted bilirubin as a major component, and black stones differed from brown pigmented stones by the presence of Ca palmitate. Bilirubin and palmitate were thermally more stable than cholesterol; hence pigmented gallstones were thermally more stable than cholesterol gallstones, as seen in TGA study. CONCLUSION: Pigmented gallstones were most common in this study of gallstones from the Jharkhand region.

The systematic classification of gallbladder stones.

BACKGROUND: To develop a method for systematic classification of gallbladder stones, analyze the clinical characteristics of each type of stone and provide a theoretical basis for the study of the formation mechanism of different types of gallbladder stones. METHODOLOGY: A total of 807 consecutive patients with gallbladder stones were enrolled and their gallstones were studied. The material composition of gallbladder stones was analyzed using Fourier Transform Infrared spectroscopy and the distribution and microstructure of material components was observed with Scanning Electron Microscopy. The composition and distribution of elements were analyzed by an X-ray energy spectrometer. Gallbladder stones were classified accordingly, and then, gender, age, medical history and BMI of patients with each type of stone were analyzed. PRINCIPAL FINDINGS: Gallbladder stones were classified into 8 types and more than ten subtypes, including cholesterol stones (297), pigment stones (217), calcium carbonate stones (139), phosphate stones (12), calcium stearate stones (9), protein stones (3), cystine stones (1) and mixed stones (129). Mixed stones were those stones with two or more than two kinds of material components and the content of each component was similar. A total of 11 subtypes of mixed stones were found in this study. Patients with cholesterol stones were mainly female between the ages of 30 and 50, with higher BMI and shorter medical history than patients with pigment stones (P<0.05), however, patients with pigment, calcium carbonate, phosphate stones were mainly male between the ages of 40 and 60. CONCLUSION: The systematic classification of gallbladder stones indicates that different types of stones have different characteristics in terms of the microstructure, elemental composition and distribution, providing an important basis for the mechanistic study of gallbladder stones.

A microstructural study of gallbladder stones using scanning electron microscopy.

OBJECTIVE: The objective of this study was to describe the microstructure of different types of gallbladder stones to better understand the basis of gallbladder stone formation. METHODS: Gallbladder stones from 387 patients with cholecystolithiasis were first analyzed by Fourier transform Infrared spectroscopy to identify the type of the gallbladder stone, and they were then examined using scanning electron microscopy to define their microstructure. RESULTS: Cholesterol stones were mainly composed of plate-like or lamellar cholesterol crystals stacked tightly in a radial, cord-like, or irregular staggered arrangement. A small number of bilirubinate particles were seen occasionally. Pigment stones were mainly composed of loosely arranged bilirubinate particles with different shapes (sphere-like, clumping-like, or amorphous). Calcium carbonate stones were composed of calcium carbonate crystals having many shapes (bulbiform, ellipsoid, fagot-shaped, fusiform, hawthorn-shaped, cuboid, button-shaped, lamellar, broken firewood-shaped, rod-shaped, acicular, or crushed sugar cane-shaped). Bulbiform shaped crystals were the most common. Phosphate stones were mainly composed of different sized echin-sphere-like or rough bulbiform crystals. Bilirubinate particles were always adherent. Calcium stearate stones usually had a network structure with adherent bilirubinate particles. Protein stones usually had a honeycomb or chrysanthemum petal-like structure. Cystine stones were composed of hexagonal cystine crystals, some of which had prominent edges. Mixed stones presented different shapes according to their mixed components. CONCLUSION: Different types of gallbladder stones had characteristic shapes and elements. This study provides an objective basis for further research regarding gallbladder stone formation.

Blackberry pigment (whitlockite) gallstones in uremic patient.

Black pigment gallstones represent nearly the 15% of all gallstones and are usually related with the typical "hyperbilirubinbilia" factors as hemolysis, ineffective erythropoiesis, pathologic enterohepatic cycling of unconjugated bilirubin, cirrhosis and with gallbladder mucosa (parietal) factors as adenomyomatosis. During a prospective study on 179 patients who underwent cholecystectomy for gallstone disease a 69-year-old female with predialysis chronic kidney disease was operated for symptomatic gallstone. The removed gallstones were black pigment gallstones, with an irregular (as small blackberry) surface. Analysis of the stones revealed a great amount of whitlockite (Ca Mg)3 (PO4)2. Recent studies on chronic renal failure patients found that chronic uremia is associated with an increased risk of gallstones formation (22%) as it seems in women affected by primary hyperparathyroidism (30%). The presence of calcium phosphate gallstones in these patients have been never described. In conclusion, further studies could be necessary to establish the role of chronic renal failure and of primary and secondary hyperparathyroidism in gallstones formation and, in particular, if dialysis and predialysis patients have an higher risk to develop cholesterol and black pigment gallstones in particular of the "blackberry" (whitlockite) subtype.

New insights into the chemical and isotopic composition of human-body biominerals. I: Cholesterol gallstones from England and Greece.

We have analyzed gallstones from four patients of Europe and particularly from England (including samples from a mother and a daughter) and Greece. According to the XRD, FTIR, NMR and laser micro-Raman results the studied materials correspond to typical cholesterol monohydrate (ChM). The micro-morphology of cholesterol microcrystals was investigated by means of SEM-EDS. The XRF results revealed that Ca is the dominant non-organic metal in all gallstones (up to ∼1.95wt.%) together with Fe, Cu, Pb and Ni (up to ~19ppm for each metal). Gallstones from England contain additional Mn (up to ~87ppm) and Zn (up to ∼6ppm) while the sample of the mother contains negligible Zn and Mn, compared to that of her daughter, but significant As (~4.5ppm). All cholesterol gallstones examined are well enriched in potentially toxic metals (Pb, as well as Ni in one case) and metalloids (As also in one case) as compared to the global average. The position of Zn, which is a characteristic biometal, in the structure of cholesterol, was investigated by molecular simulation using the Accelrys Materials Studio(®) software. On the basis of IRMS results, all gallstones examined exhibit a very light δ(13)C signature (average δ(13)C ~-24‰ PDB). Gamma-ray spectrometry measurements indicate the presence of (214)Pb and (214)Bi natural radionuclides due to the (238)U series as well as an additional amount of (40)K.

Fixed-point and stratified analysis of the fine structure and composition of five gallstones with Fourier transform infrared (FT-IR) specular reflection spectroscopy.

By investigating the identities, amounts, and distributions of the major components of human gallstones, the formation mechanism of calculi may be elucidated. Using FT-IR specular reflection spectroscopy, the compositions of five common types of gallstones, including cholesterol calculus, bile pigment calculus, cholesterol-bilirubin calculus, mixed calculus, and black stones, were positionally, qualitatively, and quantitatively analyzed. Several organic and inorganic substances were found in human gallstones, including cholesterol, calcium bilirubinate, calcium carbonate, calcium phosphate, and calcium stearate. Among them, cholesterol and calcium bilirubinate were the main components. The amounts of these substances varied in different types of gallstone. The distributions of cholesterol and calcium salts (with the exception of calcium bilirubinate) were also closely associated with location. In the various calculi, the amounts of cholesterol and calcium bilirubinate in the cores, the layered structures outside the cores, and the outer shells were higher than those of other substances. The identities of the major components of calculi were similar in different types of gallstones, but their amounts and distributions were significantly different. The analytical method used in this study, which was able to accurately determine the location, quality, and quantity of the major components of gallstones, would be an effective tool to study the formation mechanism of calculi.

An animal model of black pigment gallstones caused by nanobacteria.

Black pigment gallstones are often founded in gallbladder, but their pathogenesis is unclear. The present study was undertaken to study the role of nanobacteria in pigment gallstone formation in Japanese white rabbits. Nanobacteria were successfully cultured from 3 of 7 cholecystolithiasis patients gallbladder (without acute cholecystitis) bile samples and affirmed by Hoechst 33258 staining and specific immunostaining using monoclonal antibody. Nanobacteria were injected into rabbits' gallbladder. After 2 weeks follow-up, the incidence of black pigment gallstones in rabbits was significantly greater in nanobacteria-injected group (8/10) than that in DMEM-injected group (2/10) and that in hydroxyapatite-injected group (2/10). This study indicates that nanobacteria exists in gallbladder bile of cholelithiasis patients and causes the formation of black pigment gallstones in rabbits. For the first time, we successfully established an animal model of black pigment gallstones caused by nanobacteria. This will be helpful in further delineating the pathogenesis of black pigment gallstones.

Bacteriological and electron microscopic examination of primary intrahepatic stones.

BACKGROUND: Primary intrahepatic cholelithiasis is usually combined with biliary tract infection. This research was undertaken to investigate the relationship between intrahepatic stones and biliary tract infection. METHODS: Thirty-five bile samples and 30 stones specimens were cultured for bacteria and 12 stones specimens were examined with a scan electron microscope (SEM) or a transmission electron microscope (TEM). RESULT: 94.2% bile samples and 96.7% stones specimens were positive in bacteria culture. Bacteria were found in stones under SEM and TEM. CONCLUSION: Bacteria in stones are associated with the infection of the biliary tract.

Effects of hydrophobic and hydrophilic bile salts on gallstone growth and dissolution in model biles.

UNLABELLED: Cholesterol crystallization is a prerequisite for gallstone formation and growth, whereas dissolution of crystallized cholesterol forms the basis of nonsurgical therapy. Crystallization has been studied in detail, but dissolution mechanisms and effects of gallstones are largely unknown. METHODS: We evaluated gallstone growth or dissolution, cholesterol crystallization and lipid distribution into various phases, in model biles with low or intermediate phospholipid contents (crystal-containing left two-phase or central three-phase zones), and with high phospholipid or low cholesterol contents (crystal-free right two-phase or bottom one-phase zones). RESULTS: In model biles with added gallstones plotting in left two-phase and central three-phase zones, gallstone masses increased, whereas crystallization in the aqueous phase was less than without gallstones (P<0.001). In biles plotting in the right two-phase zone, gallstone masses decreased, depending on bile salt hydrophobicity (TUDC>TC>TCDC: P<0.001). In biles plotting in the bottom one-phase zone containing TC or TCDC, gallstone masses increased. In contrast, gallstone masses decreased in case of TUDC with preferential distribution of cholesterol into emerging vesicles. CONCLUSIONS: Our findings suggest competition between gallstone surface and surrounding aqueous phase for precipitation of cholesterol in crystal-containing zones. Different gallstone dissolution mechanisms may exist for TUDC and TCDC.

Structural and analytical comparison of gallbladder stones collected from a single patient: studies of five cases.

We observed the gross and fine structure of gallbladder stones collected from five adult patients (cases I-V) by optical photography, radiography, scanning electron microscopy, and backscattered electron microscopy, and then measured the components by energy-dispersive X-ray microanalysis and infrared spectroscopy. From the stones, calcium (Ca) phosphate, Ca bilirubinate, and Ca palmitate or fatty acid Ca were identified. The 3 cholesterol stones (case I) and the 2 brown pigment stones (case II) showed macroscopic homogeneity, respectively. In addition, their fine structure and components were also similar to each other. The black pigment stones (case III) showed macroscopic homogeneity, but they were divided into radiopaque (approximately 30 stones) and radiolucent types (approximately 60 stones). The former had Ca phosphate in the center surrounded with Ca bilirubinate, and the latter was dotted with minute deposits of Ca bilirubinate. The 6 cholesterol stones (case IV) were divided into two types in size. The 5 large stones, of macroscopic homogeneity, had a core region of Ca palmitate and clear concentric rings of Ca phosphate, whereas the smaller stone was almost filled with Ca phosphate deposits in the center. From the different distributions of Ca phosphate, the smaller stone may have been formed later than the 5 large stones. Case V contained 4 stones. The 3 large cholesterol stones, of more or less macroscopic homogeneity, had a core region and concentric rings of Ca phosphate, but 1 smaller stone was dotted with minute deposits mainly containing iron (Fe) and/or silicon elements (rare type). Therefore, the stones of cases III, IV, and V showed considerable heterogeneity, respectively. In many stones, the initial precipitation of Ca salts will have become the nidus, and the concentric rings and dotted deposits of Ca salts may have accelerated cholesterol stone growth. In addition, the dotted deposits of Ca bilirubinate in the black pigment stones and the dotted deposits containing Fe in the rare stone may have become also the nidi.

Rokitansky-Aschoff sinuses of the gallbladder are associated with black pigment gallstone formation: a scanning electron microscopy study.

Rokitansky-Aschoff sinuses are the result of hyperplasia and herniation of epithelial cells through the fibromuscular layer of the gallbladder wall and are usually referred to as adenomyomatosis. The role of this study is to demonstrate that Rokitansky-Aschoff sinuses of the gallbladder are a risk factor for the formation of black pigment gallstones. A total of 179 removed gallbladders, were hystologically examined. Sixty-four of the 179 consecutive cholecystomized patients had typical adenomyomatosis. Thirty-eight of the 64 patients with adenomyomatosis had black pigment gallstones, alone (n=22) or in association with single (n=12) or multiple (n=4) cholesterol gallstones in the same gallbladder. Twelve of these patients did not have the typical risk factors for black stones (hemolysis, cirrhoses, gastrectomy, etc). Gallstones were examined by infrared spectroscopy and X-ray diffractometry. In addition, in a subset of 14 patients, the gallstones and the gallbladder wall were examined by scanning electron microscopy. At least in the initial phases of formation, Rokitansky-Aschoff sinuses were found close to small intraparietal vessels and sometimes they contained black pigment microstones. After the fourth to fifth decades of life, black gallstones can be found in the Rokitansky-Aschoff sinuses and in the main gallbladder lumen. Black pigment gallstones can form in Rokitansky-Aschoff sinuses of the gallbladder in absence of the typical risk factors for bilirubin suprasaturation of bile.

Biochemical and physicochemical changes in collagen isolated from the gall bladder of gall stone patients.

Abnormal gall bladder motor function with delayed emptying and stasis are the contributory factors of gall stone formation. Since collagen is the major contractile protein, this study was designed to find out whether the biochemical and physicochemical changes of collagen contribute to the pathogenesis of gall stone formation. Collagen was isolated from the gall bladder of 25 gall stone patients undergoing cholecystectomy and from that of 20 gall stone free subjects. The levels of total, soluble and insoluble collagen were determined. The activity levels of collagenase (3.4.23.3) and protease (3.4.24.11) were assessed. Levels of susceptibility of collagen to denaturing agents 2 M potassium thiocyanate and 8 M urea were estimated. Aldehyde content, shrinkage temperature and tensile strength were also determined in isolated collagen. SDS-PAGE was carried out and alpha, beta fractions were quantified. The total and insoluble collagen contents were significantly high in gall stone patients. The activity levels of collagenase and protease were significantly low. Elevated levels of lipid peroxides, aldehyde content, shrinkage temperature and tensile strength were observed in gall stone patients. There is a significant elevation in the beta fraction and a decrease in alpha/beta ratio. Ultramicroscopic structure of gall bladder revealed derangement of collagen fibres and altered tissue architecture. The results showed that the qualitative and quantitative alterations in collagen also contribute for the defective contractility and stasis of gall bladder in gall stone patients.

[Study of the insoluble remainder of human gallstones by scanning electron microscope and Fourier transform infrared spectrum].

The insoluble remainder of brown pigment and mixed gallstones which were dissolved with chloroform, ethanol, ether, and hydrochloric acid were studied by Fourier transform infrared (FT-IR) spectrum and scanning electron microscope. It was found that bilirubinate salts, proteins, fatty salts, phosphate and calcium carbonate are the main components in the insoluble remainder of gallstones. The secondary structure of proteins in brown pigment gallstones may be predominated by alpha-helix. The microstructure of proteins in brown pigment stones is membranous, phosphate and bilirubinate salts were trapped within the membranes. The relationship between the insoluble components and formation of gallstones was discussed.

[Clinical and pathogenetic significance of biliary tract abnormalities in a child with cholelithiasis]. / Kliniko-patogeneticheskoe znachenie anomalii zhelchnovyvodiashchikh putei u detei s kholelitiazom.

The clinical peculiarities of cholelithiasis in the children with biliary tract's anomalities are presented in this paper. The data of clinical, instrumental examinations and anamnesis are analysed. Although, there are some clinical peculiarities of cholelithiasis in the patients with biliary tract's anomalities, intrascopic methods of examination are preferable. Ultrasonography is used as quick method during before hospital examination. Retrogdade cholangiopancreatography in special hospital is the main diagnostic method in such patients, that defines the date, volume and kind of operation.

Participacion bacteriana en estructura y composicion de cálculos biliares / Bacterial participation in the structure and composition of the gallstones

Se estudió el comportamiento de cálculos biliares en líquidos de diferente densidad incluyendo bilis hepática y bilis vesicular. Curiosamente cálculos de características similares en forma, volumen, peso y densidad tienen diferente localización porque alguns flotan en la superficie y otros se introducen hasta el fondo de las soluciones. Ello, debido a que en su estructura interna se encontraron espacios vacíos únicos o múltiples y pueden clasificarse como "cálculos flotadores" o "ballon calculli" . Estos espacios estarían ocupados por gases de origen bacteriano que fueron atrapados durante los processos de cristalización y estructuración de cálculos. Desde el punto de vista químico se relevó que en su composición existe lisolectina, cuya presencia se explica por actividad enzimática de fosfolipasa de origen bacteriano.

Bacteriological and electron microscopy examination of brown pigment stones.

Bacteriological and morphological studies of 38 brown pigment common duct stones were performed. Stone cultures were positive for bacteria in 80.5% of those studied. Enterococci were the most common organisms that were isolated. Scanning electron microscopy showed the presence of bacteria in 84.2% of the stones. The bacteria were seen embedded within an amorphous matrix in alternating layers of flakelike crystals. Transmission electron microscopy showed the presence of gram-positive and gram-negative bacteria surrounded by a ruthenium red-stained exopolysaccharide material. Results of the bacteriological and morphological studies confirmed the close relationship between the presence of bacteria and the development of brown pigment stones.