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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 3  |  Page : 328-331
Significance of cytomorphological and microbiological examination of bile collected by endoscopic cannulation of the papilla of vater


1 Department of Pathology, Moti Lal Nehru Medical College, Allahabad, India
2 Department of Gastroenterology, Moti Lal Nehru Medical College, Allahabad, India

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Date of Web Publication12-Aug-2009
 

   Abstract 

Background: Bile analysis yields important information such as "biliary microlithiasis" and biliary tract colonization by microorganisms like Salmonella typhi or paratyphi, Escherichia coli , etc., which may progresses to cholelithiasis and have been found to have a role in the development of gallbladder cancer in India. Aim: To perform microscopic, cytomorphological and microbiological examination of bile collected during endoscopic retrograde cholangiopancreatography in patients with benign and malignant lesions of the gallbladder. Materials and Methods: Bile was collected from 48 patients by a catheter inserted through the cystic duct during endoscopic retrograde cholangiopancreatography. Direct microscopy and grading of crystals was performed. Smears prepared from centrifuged deposits were stained with Giemsa, Papanicolaou stain and Gram's stain for cytological and microbiological examination. Special staining for Helicobacter pylori was performed using Loeffler's Methylene blue and Warthin Starry stain. The rest of the sample was used for culture and complete microbiological examination. Results: Thirty-six patients had inflammatory lesions while 12 had malignant lesions. Crystals were observed in 93% of the cases, 13 (28.8%) had only cholesterol crystals, three (6.6%) had bilirubinate and 29 (64.4%) had both cholesterol and bilirubinate crystals. Smears from the centrifuged deposit mainly showed coccoid or cocobacillary bacteria on Gram's staining (81.3%). Five of 12 (41.6%) malignant cases showed epithelial atypia while none of the benign or inflammatory lesions showed such a change in hematoxylin and eosin and Pap-stained smears. Microbiologic analysis showed Staphylococcus aureus (14%), S. saprophyticus (5.5%), Peptococcus (5.5%), Peptostreptococcus (5.5%), Proteus mirabilis (5.5%), E. coli (17%), Enteorbacter cloacae (5.5%) and H. pylori (2.8%). Conclusion: Complete microscopic and microbiological examination of bile directly obtained from the common bile duct during endoscopic retrograde cholangiopancreatography yielded good results as fresh samples could be analyzed. It may help in diagnosis of many gallbladder lesions in early stages, thereby reducing the morbidity and mortality.

Keywords: Bile, bile crystals, endoscopic retrograde cholangiopancreatography, gallbladder, gallstones

How to cite this article:
Misra V, Misra SP, Singh PA, Dwivedi M, Verma K, Narayan U. Significance of cytomorphological and microbiological examination of bile collected by endoscopic cannulation of the papilla of vater. Indian J Pathol Microbiol 2009;52:328-31

How to cite this URL:
Misra V, Misra SP, Singh PA, Dwivedi M, Verma K, Narayan U. Significance of cytomorphological and microbiological examination of bile collected by endoscopic cannulation of the papilla of vater. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 Dec 6];52:328-31. Available from: http://www.ijpmonline.org/text.asp?2009/52/3/328/54986



   Introduction Top


Bile analysis yields important information such as "biliary microlithiasis," which may be associated with biliary colic, cholelithiasis and pancreatitis. [1] Microlithiasis progresses to cholelithiasis, which is a well-established predisposing factor for carcinoma of the gallbladder (Ca GB). [2] Microbiological studies conducted on bile have revealed a varied bacterial microflora. Recent studies have shown the presence of anti-Helicobacter pylori antibodies, bacterial antigens and genomic material in the bile. [3] In an earlier study from our center, H. pylori has been demonstrated in areas of gastric metaplasia and its DNA was isolated from the gallstones of the same patients. [4] Biliary tract colonization by microorganisms like  Salmonella More Details typhi or paratyphi,  Escherichia More Details coli , etc. has been found to have a role in the development of gallbladder cancer in India. [5]

A detailed cytomorphological study of the bile has been attempted in the past and has shown metaplastic and dysplastic changes in epithelial cells, which may act as a precursor to neoplastic lesions of the gallbladder. [6] Demographic data show that cholelithiasis and Ca GB have been frequently observed in North India, especially in the Gangetic belt; hence, there is a need for early detection of risk factors that can lead to the development of (Ca GB), with particular stress on the role of microlithiasis and microbiological flora of the bile. [7]

As there were no studies from this part of the country describing in detail microscopic, microbiological and cytomorphological features of the bile, the present study was undertaken with the aim of performing a complete examination of bile collected during endoscopic retrograde cholangio pancreatography and to determine its clinical significance.


   Materials and Methods Top


Bile was collected from 48 cases by a catheter inserted through the cystic duct during ERCP performed for various conditions [Table 1]. Bile was collected in a sterilized test tube. After centrifugation, a part of it was put to direct microscopy and type and quantity of crystals were noted. The grading of crystals was performed according to the Junifer and Barson grading system [8] as follows:

Grade I: <10 crystals/Low power field

Grade II: 10-15 crystals/Low power field

Grade III: >25 crystals/Low power field

Grade IV: >1/High power field or microliths

A positive result for cholesterol crystals was taken if grade 1-4 (i.e., any crystal) was present and, for calcium bilirubinate crystals, as grade 3-4, i.e. when >25 crystals/LPF or >1 crystal/ HPF was present. Cytological examination was performed in the smears made from the deposits. Smears were stained by Leishman-Giemsa, hematoxylin and eosin (H and E), Papanicolaou and Gram's stain.

Special staining for H. pylori was performed using Loeffler's Methylene blue [9] and Warthin Starry stain. The rest of the sample was used for complete microbiological examination, which included gram staining, culture on routine and special media and biochemical reactions to identify the exact nature of the organism.


   Observations Top


Forty-eight bile samples were obtained by endoscopic retrograde cholangiopancreatography. The clinicoradiological diagnosis of these cases was as shown in [Table 1]. Thirty-six patients presented with inflammatory lesions, with mean age of 44 21.3 (range 22-65) years while 12 had malignant lesions, with mean age of 54 18.6 (range 39-79) years. Inflammatory lesions mainly included cholelithiasis (29/36) and choledocholithiasis with cholelithiasis (5/36). One sample each was from patients with acute cholangitis and duodenal ulcer with obstructive jaundice. Nine samples were obtained from patients with malignant lesions of gallbladder while three were from patients with periampullary tumor.

The results of direct microscopy findings are summarized in [Table 2]. On direct microscopy, crystals were observed in 45 (93%) cases. Cholesterol crystals were recognized as rectangular or rhomboid plates with notched corners. Bilirubinate crystals occurred as amorphous crystals, clumps of fine needles or as rhombic plates [Figure 1] and [Figure 2]. On direct microscopy, 13 (28.8%) samples had only cholesterol crystals (>4/HPF), which according to the classification by Juniper and Burson, was grade IV. Ten of 13 samples showed clumps of cholesterol crystals forming microliths [Figure 1]. Only bilirubinate crystals were found in three (6.6%) cases, 29 (64.4%) cases had both cholesterol and bilirubinate crystals. The cells seen were normal epithelial cells in 24 cases (50%), red blood cells in three cases (6%) and pus cells in 19 cases (39.5%). Epithelial cells with dysplasia were seen in five cases (10.4%).

The slides prepared from centrifuged deposits of bile and stained with H and E, Grams stain, Methylene blue and Warthin starry stain could be examined successfully in 32 of 45 cases (71%). Twenty-six of 32 (81.3) specimens showed coccoid or cocobacillary bacteria on Gram's staining [Figure 3]. The morphology of epithelial cells with and without atypia could be further identified in HandE and Papanicolaou stains. All the five cases showing dysplastic epithelial cells were confirmed to have Ca GB, while none of the benign or inflammatory lesions showed such a change [Figure 4] and [Figure 5].

Only 36 bile samples could be put to complete microbiological study, including gram stain, culture on adequate culture media and biochemical reactions due to some technical problems. The results were as shown in [Table 3]. Overall microbiological and biochemical analysis revealed the following organisms: E. coli (17%), Staphylococcus aureus (14%), S. saprophyticus (5.5%), Peptococcus (5.5%), Peptostreptococcus (5.5%),  Proteus mirabilis Scientific Name Search  (5.5%) and Enteorbacter cloacae (5.5%). Organisms resembling H. pylori were found in one (2.8%) bile sample and further confirmed by positive reaction for catalase, oxidase and urease.


   Discussion Top


The present study comprising of complete bile examination collected at endoscopic retrograde cholangiopancreatography from the common bile duct in 48 patients was carried out in a population dwelling in Northern India, especially in the Gangetic belt. This region has the highest incidence of Ca GB in India. Hence, the importance of recognizing the risk factors associated or predisposing to Ca GB in this region could be further highlighted with this study. [7],[9]

Bile is usually collected from the bile duct or the lumen of the duodenum, often after stimulating the gallbladder to contract. Complete bile examination using samples directly obtained from the common bile duct during endoscopic retrograde cholangiopancreatography yielded good results as fresh samples could be analyzed. [10] The present study also underlines the numerous advantages of bile collection during endoscopic retrograde cholangiopancreatography, i.e. bile is collected during a common endoscopic retrograde cholangiopancreatography procedure performed for diagnosis and, sometimes, treatment of biliary disease. It does not require additional equipment and the bile collected yields results comparable with other standard methods of bile collection. In earlier reports, results of bile obtained during ERCP were found to be as good as those obtained from gallbladder bile. [9],[10]

Studies have shown a higher cholesterol content and lower bilirubin content in stones from North India as compared with those from South India. [11] An earlier study from our center showed gallstones to be either pure cholesterol or cholesterol-predominant mixed stones by performing chemical analysis. No pure pigment stones were seen. [4] Jayanthi et al. reported a higher incidence of pigment stones in South India. [12] The present study also showed only cholesterol crystals in a larger number of the bile samples than bilirubinate crystals.

A significantly higher incidence of Ca GB has been observed in patients harboring gallstones. [2] Cholesterol stones, particularly of large size (>3 mm), are found to be intimately associated with Ca GB. [13] Hence, there could be a significant correlation between the findings of cholesterol gallstones in North India as well as a higher incidence of Ca GB in this region. Bile analysis in the present study detected cholesterol crystals and tiny microliths that were too minute for ultrasonography or cholecystographic detection. These crystals and microliths represent stage III and stage IV, respectively, of Juniper and Burson's grading [8] and can progress to clinical cholelithiasis. [14] A recent study on bile microscopy by Saraswat et al. [1] in patients with idiopathic recurrent pancreatitis and biliary colic found a higher incidence of microlithiasis with cholesterol crystals as the cause. [1]

It has been suggested that bacteria play a role in the development of Ca GB by producing a carcinogen. A study where microbiological study of bile was performed in cases of cholecystitis showed the following results: the most common organism was E. coli , followed by Klebsiella, Pseudomonas, S. aureus, Salmonella and Bacteroids fragalis . [3] Chronic carriers of S. typhi / paratyphi also seem to be at an increased risk of Ca GB. [15] In a recent study, it was found that patients with complicated gallstone disease frequently exhibit bactibilia whereas patients with uncomplicated cholelithiasis have aseptic bile. Gram negative enteric aerobes such as E. coli , Klebsiella spp. and Proteus spp. are more commonly isolated from infected bile while Pseudomonas aeruginosa , B. fragilis and Enterococcus faecalis are less commonly cultured. [16] Another study performed by Jayanthi et al. [12] on bacterial pathogens in bile showed that a majority of the patients harboring pigment gallstones showed sterile bile whereas 50% of those with cholesterol stones had growth of bacteria. The organism isolated in mixed and pigment stones were E. coli , Klebsiella, Streptococcus fecalis and Pseudomonas while in patients with cholesterol stones, S. epidermidis , Klebsiella, S. aureus and Streptococcus fecalis were isolated. In the present study, segregation into groups according to type of gallstone detected was not performed. However, the bacterial flora consisted of S. aureus, Peptostreptocci and E. coli in 11% each and S. saprophyticus, Peptococci , Pseudomirabilis, Enterobacteriacae and H. pylori in 5.5% of the cases, each out of 18 cases, whereas 39% of the cases yielded a sterile bile culture. Recent studies have implicated H. pylori in gallstone formation. In an earlier study, we isolated H. pylori DNA from gallstones, suggesting its role in the formation of cholesterol stones. [4]

Observation of epithelial cells with dysplasia in 10.4% of bile samples on direct examination of the Giemsa-stained smear further stresses the importance of bile examination in early detection of cancer. This may help in reducing the morbidity and mortality due to Ca GB. Application of special techniques like cytochemistry or proliferation markers like AgNOR may further increase the sensitivity for detection of malignancy. [17],[18],[19]

To conclude, microscopic and microbiological examination of bile collected during ERCP may help in clinching many gallbladder abnormalities before they are clinically or radiologically evident. Early treatment and management may help in reducing the morbidity and mortality due to gallstones and associated changes.

 
   References Top

1.Saraswat VA, Sharma BC, Agarwal DK, Kumar R, Negi TS, Tandon RK. Biliary microlithiasis in patients with idiopathic acute pancreatitis and unexplained biliary pain: r0 esponse to therapy. J Gastroenterol Hepatol 2004;19:1206-11.  Back to cited text no. 1    
2.Diehl AK. Epidemiology of gall bladder cancer. A synthesis of recent data. J Natl Cancer Inst 1980;65:1209-14.  Back to cited text no. 2    
3.Sattar I, Aziz A, Rasul S. Frequency of infection in cholelithiasis. J Coll Physicians Surg Pak 2007;17:48-50.  Back to cited text no. 3    
4.Misra V, Misra SP, Dwivedi M, Shouchi Y, Dharne M,Singh PA. Helicobacter pylori in areas of gastric metaplasia in gall bladder and isolation of H.pylori DNA from gall stones. Pathology 2007;39:419-24.  Back to cited text no. 4    
5.Nath G, Singh H, Shukla VK. Chronic typhoid carriage and carcinoma of the gall bladder. Eur J Cancer Prev 1997;6:557-9.  Back to cited text no. 5  [PUBMED]  
6.Kuwabara H, Uda H. Small cell carcinoma of the gall bladder with intestinal metaplastic epithelium. Pathol Int 1998;48:303-6.  Back to cited text no. 6  [PUBMED]  
7.Dhir V, Mohandas KM. Epidemiology of digestive tract cancers in India.IV. Gall bladder and pancreas. Indian J Gastroenterol 1999;18:24-8.  Back to cited text no. 7    
8.Juniper K Jr, Burson EN Jr. Biliary tract studies II: The significance of biliary crystals. Gastroenterol 1957;32:175-208.  Back to cited text no. 8    
9.Misra V, Misra SP, Dwivedi M, Gupta SC. The Loeffler's Methylene blue stain: An inexpensive and rapid method for detection of Helicobacter pylori . J Gastroenterol Hepatol 1994;9:512-3.  Back to cited text no. 9    
10.Delchier 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;6:118-22.  Back to cited text no. 10  [PUBMED]  
11.Buscail 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;37:116-20.  Back to cited text no. 11  [PUBMED]  
12.Purwar V, Chaturvedi PK, Raju RV, Augustine P, Tandon RK. Biophysical, biochemical and morphological comparative study on bile duct stones of north and south Indian origin. Indian J Gastroenterol 1989;8:A8.  Back to cited text no. 12    
13.Jayanthi V, Pavithra A, Rao U. Gall stones and bile culture in patients with gall stone disease. Indian J Gastroenterol 1996;15:A103.  Back to cited text no. 13    
14.Kaushik SP. Current perspectives in gall bladder carcinoma. J Gastroenterol Hepatol 2001;16:848-54.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]
15.Caygill CP, Hill MJ, Braddick M. Cancer mortality in chronic typhoid and paratyphoid carrier state. Lancet 1994;343:83-4.  Back to cited text no. 15    
16.Morris-Stiff GJ, O'Donohue P, Ogunbiyi S, Sheridan WG. Microbiological assessment of bile during cholecystectomy: i0 s all bile infected? HPB (Oxford) 2007;9:225-8.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.Nath G, Singh H, Shukla VK. Chronic typhoid carriage and carcinoma of the gall baladder. Eur J Cancer Prev 1997;6:557-9.  Back to cited text no. 17  [PUBMED]  
18.Misra V, Misra SP, Dwivedi M, Gupta SC. AgNORs in diagnosis of early malignant lesions of gall bladder. Indian J Pathol and Microbiol 1995;38:383-8.  Back to cited text no. 18    
19.Gupta SC, Misra V, Singh PA, Roy Anu, Misra SP, Gupta AK. Gall stones and Carcinoma Gall Bladder. Indian J Pathol and Microbiol 2000;43:147-54.  Back to cited text no. 19    

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Correspondence Address:
Vatsala Misra
Department of Pathology, Moti Lal Nehru Medical College, Allahabad - 211 001
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.54986

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  [Table 1], [Table 2], [Table 3]

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