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Endoscopic biliary drainage
van Berkel, A.M.
Publication date
2003
Link to publication
Citation for published version (APA):
van Berkel, A. M. (2003). Endoscopic biliary drainage.
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MechanismsMechanisms of biliary stent clogging:
confocalconfocal laser scanning and
scanningscanning electron microscopy
A.M.. van Berkel', J. van Marie2, A.K. Groen', MJ. Bruno1 Departmentt of Gastroenterology and Hepatology' andd Department of Electron Microscopy'
Academicc Medical Center, Amsterdam, The Netherlands
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M E C H A N I S MM OF BILIARY STENT C L O G G I N G 133
ABSTRACT T
Introduction:Introduction: In order to compare early events in biliary stent clogging and identify
distributionn of bacteria in unblocked stents, we performed Confocal Laser Scanning (CLS)) and Scanning Electron Microscopy (SEM) on two different stent materials, polyethylenee (PE) and hydrophilic polymer coated polyurethane (HCPC).
Methods:Methods: Ten consecutive patients with postoperative benign biliary strictures were
includedd in the study. Two 10 Fr 9 cm stents, one PE and one HCPC, were inserted. Stentss were electively exchanged after 3 months and examined by CLS and SEM.
Results:Results: No differences between the two types of stents were seen. The inner stent
surfacee was covered by a uniform amorphous layer. On top ot this layer a biofilm of livingg and dead bacteria was found which in most cases was unstructured. The lumenn was filled with free floating colonies of bacteria and crystals surrounded by movablee laminar structures of mucous. In all stents an open network of large dietary fibress was seen.
Conclusion:Conclusion: In both PE and HCPC stents the same early clogging events occurred.
Thee most remarkable observation was the identification of networks of large dietary fibress resulting from duodenal reflux acting as a filter. This seems an uniform mech-anismm responsible for stent clogging.
I N T R O D U C T I O N N
Endoscopicc insertion of plastic biliary endoprostheses is a well established treatment forr obstructive jaundice. The major limitation of this technique is late stent occlu-sionn after a median duration of 3-6 months which necessitates stent exchange (1,2}. Stentt obstruction is caused by biliary sludge which consists of crystals of calcium bilirubinatee and calcium palmitate, as well as proteins, mucopolysaccharides, choles-teroll crystals and bacteria (3,4).
Itt is generally assumed that the initial event in stent blockage is adherence of pro-teinss and bacteria to the inner wall of the stent forming a biofilm. Subsequently, bil-iaryy components such as calciumbilirubinate and calcium fatty acid soaps precipitate becausee bacteria secrete Jl-glucuronidase and phosopholipases (5-7). Probably the bacteriaa are already introduced during transpapillary placement of the stent. Additionally,, after the stent is in place, bacteria can colonize the biliary tract due to refluxx from the duodenum.
Thee attachment of bacteria to the inner wall of the stent may depend on polymeric sur-facee properties (8). Different materials have been used for stent construction: polyeth-ylene,, polyurethane and teflon. In vitro studies have found a direct relation between the frictionn coefficient and the amount of encrusted material (5,9). Teflon has the lowest frictionn coefficient and therefore the maximum potential for preventing stent clogging. AA hydrophilic polymer coating was demonstrated to be effective in reducing bacterial adherencee in in vitro studies {10). The hydromer stent not only has a smooth texture butt also a coating that absorbs water and provides a hydrophilic sheath. Because
bac-1344 ENDOSCOPIC BILIARY DRAINAGE
teriaa initially attach by hydrophobic interactions, this coating might decrease bacteri-all adhesion and therefore increase stent patency. However, the positive results of dif-ferentt in vitro studies could not be confirmed in prospective clinical trials {11-14). Inn this study we performed Confocal Laser Scanning (CLS) and Scanning Electron Microscopyy (SEM) in two different stent materials, polyethylene and hydrophilic polymerr coated polyurethane, in order to compare early events in stent clogging and identifyy distribution of (living and dead) bacteria in unblocked biliary stents.
MATERIALSS EN M E T H O D S
Fromm February 2002 to December 2002, 10 consecutive patients with postoperative benignn biliary strictures were included in the study. There were 5 men and 5 women withh a mean age of 50 years (range 30*64 years). Two 10 Fr 9 cm stents were insert-edd in patients with a postoperative bile duct stenosis: one standard polyethylene stent (PE)) (Wilson Cook, Winston-Salem, N.C) and one hydrophilic polymer coated polyurethanee stent (HCPC) (Biosearch, Sommerville, N.J). The HCPC stent was soakedd in water for 5 minutes before use. Elective stent exchange was performed, as aa standard treatment, after 3 months to avoid cholangitis by clogging. None of the patientss received prophylactic antibiotics. The stents were then examined by CLS withinn 30 minutes after stent removal and by SEM.
ConfocalConfocal laser scanning microscopy
Smalll rings were cut from the stents: at the distal end, in the centre and at the prox-imall end of the stent. Specimens were stained for 15 min with SYTO 9 and propidi-umm iodide as live/dead stain, rinsed in buffer and imaged. The live/dead viability test wee used relies on the fact that membranes of dead cells are permeable to many dyes thatt cannot cross them in the living state. SYTO 9 is a very effective stain with min-imumm non-specific binding during staining of complex communities (15). For imag-ingg a Leica SP2 confocal microscope was used. Excitation was done with the 488 nm linee of the Argon-Ion laser. SYTO 9 was detected between 500 nm and 535 nm and propidiumm iodide was detected between 650 nm and 700 nm. For additional struc-turall information the reflected image of the 488 nm line was acquired.
Imagess were acquired in a 512*512 format (8 bit). A HCL PL APO 20.0 x 0.70 Imm/Corrr UV objective was used and a zoom factor between 1 and 5. The pinhole wass set at a diameter of 1 airy disc corresponding with a z-resolution of approximate-lyy 3 um. Data stacks were generated over a depth of up to 120 um with a step size of 22 um. Using the Leica software, images of SYTO 9 and propidium iodide were mergedd and stereo pairs were generated from the 3D image stacks.
ScanningScanning electron microscopy
Thee specimen were fixed in McDowell's fixative for at least 48 hours, dehydrated and finallyy dried with hexamethyldisilazane. The dried specimen were mounted on stubs
M E C H A N I S MM OF BILIARY STENT CLOGGING 135
andd coated with approximately 10 nm gold. Specimens were imaged with a Philips SEMM 525 operated at either 5 or 10 kV and equipped with an Orion frame grabber.
RESULTS S
Thee endoprostheses of each group were analyzed using both Confocal Laser Scanningg and Scanning Electron Microscopy. No patient presented with symptoms off stent clogging and no patient received prophylactic antibiotics. All analyzed stents weree patent as judged by eye. There was no difference in the amount and distribu-tionn of sludge and bacteria between polyethylene and hydrophilic coated pulyurethanee stents on both CLS and SEM.
ConfocalConfocal laser scanning microscopy
Bothh polyethylene and hydrophilic polymer coated polyurethane stents showed a similarr phenomenon. The layer attached to the internal wall of the stent was a high-lyy reflective amorphous layer (thickness approximately 15-30 mm) which for each stentt was extremely uniform in thickness and distribution. This layer formed the substratumm for the attached bacteria.
Inn some cases a structured biofilm was observed, with dead bacteria at the top layer whichh are in direct contact with the bile while covering the underlying living cells (Figuree 1). However, in most cases this layer was without any structure: dead and liv-ingg bacterial cells were interspersed or cloudy areas with living bacteria covered by deadd bacteria were seen (Figure 2). Crystals embedded in these biofilms were found occasionally.. Especially in and around these unstructured biofilms mucoid like sheetss were found which, in many cases, extended into the lumen of the stent. The thicknesss and density of unstructured biofilms were more irregular when compared withh the structured biofilms. As far as could be observed, the biofilm attached to the stentt wall never extended more than 100 p i into the lumen of the stent. Taken into accountt the inner diameter of the stent, the decline of the luminal radius and effect onn bile flow is negligible.
Thee lumen of all stents examined was filled with free floating mixed colonies of living andd dead bacteria. The size of these colonies varied considerably, from approximately 500 um cross-section to a major part of the stent diameter (2.5 mm). These colonies weree aggregated in a substance with a slightly higher reflectance than the surround-ingg bile. Small crystals (5 um or smaller) were often found dispersed in these loosely attachedd floating colonies. If large free floating crystals were found (up to 150 um), in manyy cases they or at least their fringes were covered with a mix of living and dead bacteria. .
InIn a number of cases these free floating colonies were surrounded by laminar struc-turess (probably mucous), part of a highly movable complex network of sometimes anastomosingg structures with highly reflective surfaces. These structures sometimes weree attached to the wall of the stent. In cross section the size of these empty spaces
1366 ENDOSCOPIC BILIARY DRAINAGE
variess from 50 |u.m to over 500 um. This is best seen in the 3 dimensional picture shownn in Figure 3.
Inn all stents refluxes of dietary fibres were found. These fibres formed a more rigid tangledd network which formed a kind of filter (Figure 4).
ScanningScanning electron microscopy
Directlyy on the inner surface of both stent types, the same amorphous layer of uni-formm thickness, as observed with CLS was seen (Figure 5A). This layer is loosely attachedd to the wall of the stent. Consequently, after dehydration and drying it becomess easily detached from the stent. This layer was covered by bacteria and yeastss starting to form a biofilm (Figure 6). The shape and structure of this layer was variable.. Sometimes a clear structure could be identified with the bacteria oriented perpendicularr to the stent wall and spaces in the biofilm empty of bacteria (Figure 5C).. More often no distinct orientation was found with mucous and clusters of micro organismss dispersed.
Wee also discerned a network of amorphous material, probably mucus, in which coloniess of bacteria were embedded (Figure 7 with inset).
InIn some of the stents crystals were present, whereas in other stents they were absent. Thesee crystals were found both organised in biofilms and dispersed throughout mucouss and biomaterial (Figure 5B).
Refluxx material from the gut was found over the whole length of the stent, often cov-eredd with micro organisms.
Thee HCPC stent also showed a very porous wall of the endoprosthesis in which con-trastt additives and little holes were seen (Figure 5A), whereas the polyethylene stent showedd a solid plastic surface.
Contraryy to the results from the confocal microscopy which show a volume filled withh a highly flexible and transparent structures (in fluorescence and reflected mode),, scanning electron microscopy gives the impression that this biomaterial in whateverr shape always forms a solid obstruction.
DISCUSSION N
Thee first finding in our study, found on both CLS and SEM, was the presence of an amorphouss layer covering the stent wall which has not been described before. In ear-lierr studies mostly blocked stents were studied without the use of CLS. Investigation off patent stents has the advantage that, initial stages of clogging can be studied. Particularlyy in the final stage of occlusion when biliary flow is almost absent, signifi-cantt transformations in the clogged material may take place. Different studies report-edd about amorphous material into the stent lumen but did not report an organized layerr (6,15).
Inn this study we used both CLS and SEM to collect information from the same spec-imenn using two microscopic techniques in order to integrate these findings.
M E C H A N I S MM OF BILIARY STENT C L O G G I N G 137
Thee most important difference between CLS and SEM is that CLS allows fluores-centlyy labelled in vivo images and non invasive structural images with the possibili-tyy of three dimensional reconstructions. This enables to examine complex organized structuress like biofilms without disruption or fixation. The live/dead viability test we usedd relies on the fact that membranes of dead cells are permeable to many dyes that cannott cross them in the living state. This staining system is intended for the use withh pelagic bacteria and its role in mixed species biofilm has not been established. However,, some impression of the distribution of live and dead cells can be obtained. SYTOO 9 has been shown to be a very effective stain with minimal non-specific bind-ingg during staining of complex biofilm communities (15).
Ann interesting finding in our study was that in a number of stents, living bacteria weree attached to the stent wall covered by a layer of dead bacteria. This distribution however,, was not uniform in all stents, neither in one stent nor between the stents. Thiss finding implies that living bacteria may be protected by dead bacteria which formm a physical barrier to e.g. bacterial penetration. This may also explain the fact thatt antibiotics do not prolong stent patency in clinical trials (16).
Anotherr finding was a movable cloudy network of mucus, bacteria and crystals includingg large empty spaces which was best seen by three dimensional CLS pic-tures.. The occurrence of crystals seems to be a patient dependent factor. After an amorphouss layer on the stent surface, growth of the biofilm connects bacteria to otherr bacteria, mucus and crystals forming microcolonies which also are connected too each other in a highly flexible three dimensional networks.
Thee most remarkable observation was the presence of large numbers of plant fibres thatt have refluxed from the duodenum. In our three dimensional pictures, as well as inn the SEM pictures, we found tangled networks of fibres acting as a filter. Large fibress may suddenly obstruct the stent lumen independent of accumulation from the amountt of biliary sludge. This view is in accordance with a recent study which also reportedd about the causal role of duodenobiliary reflux and suggested change in stentt design to prevent reflux (17).
Becausee in all patients two types of stents were placed (and removed) simultaneous-ly,, we were able to study the effect of different stent materials on the formation of a biofilm.. In this study we used the standard biliary stent made of polyethylene and a hydrophilicc polymer coated polyurethane stent. In theory, the coated stent might pre-ventt bacterial adhesion because bacteria initially attach by hydrophobic interactions. However,, in this study no difference in bacterial growth between the two types of unblockedd stents could be observed. This is in accordance with clinical studies in whichh no difference in stent patency is observed (13,14).
Inn the past, many investigators have used SEM techniques for biofilm studies where fixationn may induce morphological changes. CLS can demonstrate these complex networkss in aqueous biofilms by non disruptive techniques. Both microscopic tech-niquess gave an explicit additive impression of the clogging mechanism. SEM gave a
1388 ENDOSCOPIC BILIARY DRAINAGE
veryy solid impression of the biofilm development process suggesting that bacterial growthh and biofilm formation significantly contributes to stent obstruction. However,, CLS proved that these structures are open, transparent and extremely flex-ible,, forming at most a minor obstruction to the bile flow.
Inn conclusion, in both PE and HCPC type of stents the same early clogging events occurred.. The thickness of the biofilm is so small compared to the inner diameter of thee stent that it has negligible effects on bile flow. The most remarkable observation wass the identification of networks of large dietary fibres resulting from duodenal refluxx acting as some sort of filter. This seems to be the uniform mechanism respon-siblee for stent clogging. It also explains why different stent surface materials and administrationn of antibiotics or bile salts do not have any effect on stent patency.
M E C H A N I S MM OF BILIARY STENT CLOGGING 139
stentt wall
*...iJfff '
Figuree 1. CLS showing a merged colour image of livee cells (green) and dead cells (red) in a stent. Deadd and live bacteria are in a mixed colony semi attachedd to the amorphous layer of the stent wall. Thee stent material shows a strong fluorescence. Scannedd area 750um-'75oum.
Figuree 2. CLS showing an organised biofilm attachedd to the stent wall. Dead bacteria (red) in directt contact with bile, covering the living cells (green).. Scanned area 8ooum*8oou.m.
Figuree 3. CLS showing a three dimensional stereo-graphh of mucous sheets (MS) mixed with a few coloniess of bacteria (C) and crystals (arrows). Imagee dimensions: 75oiim*75oum*iooum. Pleasee use a red-green 3-D viewer to get three-dimensionall impression.
Figuree 4. CLS showing a three dimensional stere-ographh of vegetable fibres (reflux from the duode-num)) covered with bacteria. The fibres are sur-roundedd by small floating colonies and some crys-tals.. Image dimensions: 5ooum*50oum*iooum Pleasee use a red-green 3-D viewer to get three-dimensionall impression.
1400 ENDOSCOPIC BILIARY DRAINAGE
Figuree 5A. SEM of an amorphous layer of uniform thickness directly deposited on thee stent wall. The stent is porous and contains contrast additives (Bar=2o urn). Figuree 5B. SEM showing cholesterol crystals in various size deposited on the stent walll (Bar=20 urn).
M E C H A N I S MM O F B I L I A R Y S T E N T C L O G G I N G 141
Figuree 6. SEM showing the first growth of bacteria and (in this case) yeasts on the amorphouss layer (*) on the stent wall (Bar=20 u.m ).
1422 ENDOSCOPIC BILIARY DRAINAGE
REFERENCES S
ii Huibregtse K, Tytgat GN. Palliative treatment of obstructive jaundice by transpapillary introductionn oflarge bore bile duct endoprosthesis. Gut 1982; 23(5):37i-375.
22 Davids PH, Groen AK, Rauws EA, Tytgat GN, Huibregtse K. Randomised trial of self-expandingg metal stents versus polyethylene stents for distal malignant biliary obstruction. Lancett 1992; 340(8834-8835):i488-i492.
33 Groen AK, Out T, Huibregtse K, Delzenne B, Hoek FJ, Tytgat GN. Characterization of the contentt of occluded biliary endoprostheses. Endoscopy 1987; i9(2):
57"59-44 Sung JY, Leung JW, Shaffer EA, Lam K, Costerton JW. Bacterial biofilm, brown pigment stonee and blockage of biliary stents. J Gastroenterol Hepatol 1993; 8(i):28-34.
55 Coene PP, Groen AK, Cheng J, Out MM, Tytgat GN, Huibregtse K. Clogging of biliary endoprostheses:: a new perspective. Gut 1990; 3i(8):9i3-9i7.
66 Leung JW, Ling TK, Kung JL, Vallance-Owen J. The role of bacteria in the blockage of bil-iaryy stents. Gastrointest Endosc 1988; 34(i):i9-22.
77 Speer AG, Cotton PB, Rode J, Seddon AM, Neal CR, Holton J et al. Biliary stent blockage withh bacterial biofilm. A light and electron microscopy study. Ann Intern Med 1988; io8(4):546-553. .
88 McAllister EW, Carey LC, Brady PG, Heller R, Kovacs SG. The role of polymeric surface smoothnesss of biliary stents in bacterial adherence, biofilm deposition, and stent occlu-sion.. Gastrointest Endosc 1993; 39(3):422-425.
99 Dowidar N, Kolmos HJ, Matzen P. Experimental clogging of biliary endoprostheses. Role off bacteria, endoprosthesis material, and design. Scand J Gastroenterol 1992; 27(1)77-80. 100 Jansen B, Goodman LP, Ruiten D. Bacterial adherence to hydrophilic polymer-coated
polyurethanee stents. Gastrointest Endosc 1993;
39(5):670-673-111 Binmoeller KF, Seitz U, Seifert H, Tnonke F, Sikka S, Soehendra N. The Tannenbaum stent:: a new plastic biliary stent without side holes. Am J Gastroenterol 1995; 90(io):i764-1768. .
122 van Berkel AM, Boland C, Redekop WK, Bergman JJ, Groen AK, Tytgat GN et al. A prospectivee randomized trial of Teflon versus polyethylene stents for distal malignant bil-iaryy obstruction. Endoscopy 1998; 30(8):68i-686.
133 Costamagna G, Mutignani M, Rotondano G, Cipolletta L, Ghezzo L, Foco A et al. Hydrophilicc hydromer-coated polyurethane stents versus uncoated stents in malignant biliaryy obstruction: a randomized trial. Gastrointest Endosc 2000; 5i(i):8-n.
144 van Berkel AM, Bruno MJ, Bergman JJ, Van Deventer SJ, Tytgat GN, Huibregtse K. A prospectivee randomized study of hydrophilic polymer-coated polyurethane versus polyeth-ylenee stents in distal malignant biliary obstruction. Endoscopy 2003; 35(6):478-482. 155 Lawrence J.R., Neu T.R., Swerhone G.D.W. Application of multiple parameter imaging for
thee quantification of algel, bacterial and exopolymer components of microbial biofilms. Journall of Microbiological Methods 1998; 32(3):253-26i.
166 Sung JJ, Sollano JD, Lai CW, Ismael A, Yung MY, Tumala I et al. Long-term ciprofloxacin treatmentt for the prevention of biliary stent blockage: a prospective randomized study. Am JJ Gastroenterol 1999; 94(n):3i97-320i.
177 Weickert U, Venzke T, Konig J, Janssen J, Remberger K, Greiner L. Why do bilioduodenal plasticc stents become occluded? A clinical and pathological investigation on 100 consecu-tivee patients. Endoscopy 2001; 33(9)786-790.
