absence of gadolinium deposits in the peritoneal membrane of

Nephrol Dial Transplant (2010) 25: 1329–1334doi: 10.1093/ndt/gfp664Advance Access publication 10 December 2009

Preliminary Communication

Absence of gadolinium deposits in the peritoneal membrane of patientswith encapsulating peritoneal sclerosis

Eric Goffin1,*, Josef A. Schroeder3,*, Christian Weingart4, Pierre-Yves Decleire1

and Jean-Pierre Cosyns2

1Department of Nephrology, Université Catholique de Louvain, Cliniques Universitaires St Luc, Brussels, Belgium, 2Department ofPathology, Université Catholique de Louvain, Cliniques Universitaires St Luc, Brussels, Belgium, 3Department of Pathology,Regensburg University Hospital, Regensburg, Germany and 4Department of Nephrology, Regensburg University Hospital,Regensburg, Germany

Correspondence and offprint requests to: Eric Goffin; E-mail: [email protected]*E. Goffin and J.A. Schroeder contributed equally to this work.

AbstractBackground. Encapsulating peritoneal sclerosis (EPS) isa severe complication of long-term peritoneal dialysis(PD) characterized by the development of an extensive fi-brosis of the visceral peritoneum that may eventually leadto intestinal constriction. Its cause remains elusive. Ne-phrogenic systemic fibrosis (NSF), a disabling disease thatcan follow gadolinium-based contrast injection duringmagnetic resonance imaging, is characterized by systemicfibrosis of the skin, joints, liver, heart and vessels. Affect-ed tissues are infiltrated by CD34+ and CD68+ fibro-blasts. In the present study, we tested the hypothesis thatEPS could have been triggered by a previous gadoliniuminjection.Methods. We performed histopathological analysis of theperitoneal membrane of two EPS and two control patientsall exposed to long-term PD, including immunostainingwith CD34 and CD68. The presence of gadolinium andother metals was also assessed by conventional and energy-filtered transmission electron microscopy.Results. Numerous CD34+ and CD68+ cells were foundin both the EPS and control patients within the vascularendothelium and in macrophages, respectively, but not ininterstitial fibrocytes, as it could be expected in NSF. Notrace of gadolinium deposits could be found in the fourperitoneal samples; dispersed tiny iron inclusions wereevidenced in the connective tissue of both EPS patients.Conclusions. These findings argue against the implicationof gadolinium in the development of EPS in long-term PDpatients.

Keywords: encapsulating peritoneal sclerosis; gadolinium; magneticresonance imaging; nephrogenic systemic fibrosis; peritoneal dialysis

Introduction

Encapsulating peritoneal sclerosis (EPS) is a rare but dev-astating complication of long-term peritoneal dialysis (PD).It is characterized by fibrosis of the visceral peritoneum thatmay eventually lead to extensive intestinal constriction withpersistent, intermittent or recurrent bowel obstruction,sometimes associated with bloody effluent. Weight lossand malnutrition usually ensue. Mortality is high, despitevarious treatment options [1–3]. The cause of EPS remainselusive, though multiple triggering factors have been in-criminated (long-term exposure of the peritoneal membraneto poorly biocompatible peritoneal dialysates, repeatedperitoneal infections, genetic predispositions…) [1,4]. Anincreased incidence of EPS following renal transplantation(TP) in PD patients has been suspected in the last 2–3years[5–7].

Recently, a systemic illness characterized by skin andjoint fibrosis [8], together with systemic inflammation[9–11], has been reported in end-stage renal disease andhaemodialysis patients and has been linked to a previousexposition to gadolinium-based contrast material duringmagnetic resonance imaging (MRI) [9,10]. The disorderhas been called nephrogenic systemic fibrosis (NSF). Gad-olinium, initially demonstrated in the skin of NSF patients[12–14], was subsequently found in numerous other tis-sues including the myocardium, blood vessels, skeletalmuscles, testis, liver and lungs [14–17]. In patients withchronic renal disease, the clearance of gadolinium is mark-edly delayed, allowing a process of transmetalation. Dur-ing this process, ions such as calcium, zinc, iron, copperand aluminium may substitute for gadolinium in the metalcomplex, leading to deposition of free gadolinium and ironin tissues affected by NSF [15,18]. It has been hypothe-sized that this massive free gadolinium and iron deposition

© The Author 2009. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: [email protected]

Dow

nloaded from https://academ

ic.oup.com/ndt/article-abstract/25/4/1334/1860471 by guest on 16 N

ovember 2018

induces the pro-inflammatory process that leads to NSF.The diagnosis of NSF relies on the demonstration withina deep dermal biopsy of fibrosis and specific histopatho-logic features, such as the identification of CD34+ dendrit-ic and CD68+ mononucleated cells among collagen fibres[19,20]. Gadolinium is poorly removed by conventionalPD [21] and NSF has also been described in PD patients[22–24].

In the present study, we tested the hypothesis that gado-linium could deposit within the peritoneal tissue of PD pa-tients with EPS, thus representing a localized form of NSFconfined to the peritoneal membrane, a hypothesis thatmight explain the recently observed increased incidenceof post-renal transplant EPS. Peritoneal biopsies fromtwo EPS patients and one control patient exposed to gado-linium while on PD and from one control PD patient non-exposed to gadolinium were obtained after renal TP andstudied by immunostaining and ultrastructural analysis.

Materials and methods

Clinical presentation

Patient 1. A 61-year-old male with end-stage renal failure (ESRF) second-ary to membranoproliferative glomerulonephritis received a renal cadaverTP after 100 months on PD. He had not presented any infectious perito-nitis while on PD. MRI of the aortoiliac vessels with gadolinium injection(OmniscanR, a single dose of 10mmol) had been performed in May 2005,9 months before TP. Immunosuppressive regimen included tacrolimus,azathioprine and steroids. A peritoneal biopsy, taken at the time of peri-toneal catheter removal, 2 months after TP, showed a denuded mesothe-lium and a thickened, highly vascularized submesothelial fibrous band,suggestive of EPS. Four months later, he presented with intestinal suboc-clusion. At surgery, the macroscopic appearance of the peritoneal mem-brane confirmed the diagnosis of EPS so that no specific intervention wasperformed. The symptomatology resolved with nasogastric aspiration,fasting and temporary parenteral nutrition. The steroid dosage was tran-siently increased and tamoxifen was initiated. Subsequently, the patientwas re-admitted four times for similar episodes, all resolving without sur-gery. Currently, 30months after TP, the patient is asymptomatic, thoughencasement of small-bowel loops and thickening of the intestinal wall arestill present (Figure 1). No systemic signs of NSF appeared within hisfollow-up.

Patient 2. A 67-year-old male with ESRF secondary to nephrosclerosisreceived a renal cadaver TP after 55 months on PD. He had presented asingle episode of Staphylococcus epidermidis peritonitis after 7months onPD. MRI of the aortoiliac vessels with gadolinium injection (OmniscanR,a single dose of 10mmol) had been performed in February 2005, 20months before TP. Immunosuppressive regimen included cyclosporine,azathioprine and steroids. One week after TP, he underwent an aortobife-moral replacement because of spontaneous iliac artery dissection. The in-tervention was complicated by Serratia marcescens peritonitis that hasbeen cured with meropenem. A peritoneal biopsy, taken at the time ofPD catheter removal, 5 weeks after TP, showed a denuded mesotheliumand a thickened, highly vascularized submesothelial fibrous band, sugges-tive of EPS. Eight months after TP, he presented with several bouts ofintestinal subocclusion. Abdominal CT scan disclosed peritoneal thicken-ing and several loculated fluid collections, typical of EPS. Total parenteralnutrition was initiated, together with an increase in the steroids dosage andthe prescription of tamoxifen, without improvement of the clinical condi-tion. One year later, enterolysis with 30 cm small-bowel resection wasperformed. Currently, the clinical condition of the patient is slightly im-proving. No systemic signs of NSF had appeared within his follow-up.

Control 1. A 61-year-old female with ESRF secondary to diabetic ne-phropathy received a renal cadaver TP after 31months on PD. She had notpresented any infectious peritonitis while on PD. MRI of the aortoiliacvessels with gadolinium injection (MagnevistR, a single dose of 15mmol)had been performed in September 2004, 18 months before TP. Immuno-suppressive regimen included tacrolimus, mycophenolate mofetil andsteroids. A peritoneal biopsy performed at the time of peritoneal catheter

removal, 5 weeks after TP, showed moderate signs of submesothelial fi-brosis. The patient did not develop any signs of EPS following TP, with afollow-up of 32 months.

Control 2. A 44-year-old male with ESRF secondary to IgA nephrop-athy received a renal cadaver TP after 19months on PD. He had not pre-sented any infectious peritonitis while on PD. MRI had not beenperformed prior to TP. Immunosuppressive regimen included tacrolimus,mycophenolate mofetil and steroids. A peritoneal biopsy, taken at the timeof peritoneal catheter removal, 6months after TP, was normal. Currently,35months after TP, the patient has no intestinal problems.

Histopathology

All peritoneal biopsies taken in the parietal peritoneum at the time of cath-eter removal from both EPS and control patients were fixed in 4% buff-ered formalin (pH 7.0). Paraffin sections were stained with haematoxylinand eosin (H&E) for light microscopy and processed by immunohisto-chemistry for the assessment of the cellular expression of CD34 andCD68.

Conventional electron microscopy and electron spectroscopic imaging

From a selected (positive CD34 and CD 68 evidence) paraffin block withembedded peritoneal tissue (one per patient) H&E slide guided tissuecores (three per block, diameter 2mm) were punched, deparaffinized inxylene, rehydrated with downgraded ethanols and cacodylate buffer andpost-fixed with 4% glutaraldehyde in 0.1M cacodylate buffer (pH 7.3)and subsequently with osmium tetroxide (both 1h). After being mincedinto smaller pieces, this tissue was routinely dehydrated in an automatedmanner in graded ethanols (LYNX, Leica/Vienna tissue processor) andembedded in epoxy resins (EmBed 812, Science Services/Munich). Dou-ble-stained (toluidine blue/basic fuchsine) semi-thin sections were pre-pared for relevant structure trimming for the adjacent ultramicrotomy(Ultracut S, Leica/Vienna)—two (control tissue) or three (EPS tissue)Epon blocks per punched tissue core. From the EPS patient blocks, anadditional step (100µm deeper) of semi-thin and following ultrathin sec-tions were prepared. For conventional electron microscopy examination,the produced ultrathin sections (80nm) were double stained with aqueousuranyl acetate and lead citrate and examined under the LEO912AB (CarlZeiss, Oberkochen) energy-filtered transmission electron microscope(EFTEM), operated at 100kV in electron energy zero-loss mode, allimages were acquired by a 1 × 1-k pixel side-entry digital camera.

The elemental analysis of gadolinium, calcium and iron was performedon very thin sections (approximately 40nm) without any heavy metalpost-staining using the electron energy-filtered microscope operationmode (EFTEM).

This technique provides a very high structural resolution (0.1nm) [25]and sensitivity (three to six atoms, element dependent) [26–31] for ele-ment detection and localization. It is based on the phenomenon that pri-mary beam electrons passing through a sample interact with target atoms

Fig. 1. Abdominal CT scan of Patient 1 showing encasement of small-bowel loops and thickening of the intestinal wall (arrows); R, right.

1330 E. Goffin et al.

Dow



ovember 2018

of the specimen and lose a defined, element-specific amount of energy(‘inelastic scattering’). The energy loss of the beam electrons is analysedby an in-column integrated energy filter (spectrometer); a special slit ap-erture selects electrons at the element-specific energy-loss level for imag-ing the elemental distribution in the sample.

In resin-embedded tissue specimens, two modes of EFTEM are used:electron energy-loss spectroscopy (EELS) records the whole energy-lossrange (0–2,500eV) as a complete energy spectrum where ‘edges’ at char-acteristic energy levels provide information about the chemical composi-tion of the sample. The spatial distribution of elements present in thesample can be mapped by electron spectroscopic imaging (ESI) using ex-clusively inelastically scattered electrons with the element-specific energyloss [32–38].

More technical details of the applied ESI and EELS are described else-where [14].

Results

Histopathology and immunostaining

Representative histologic changes of the parietal peritonealmembrane of Patient 1 are shown in Figure 2. H&E-stainedsections of the peritoneal tissue showed a denuded mesothe-lium and a thickened, highly vascularized submesothelial fi-brous band together with normal adjacent adipose tissue.The upper part of the submesothelial fibrous band had a fi-brohyalin appearance with low inflammatory cellular infil-tration (Figure 2A). Immunoperoxidase staining of the

peritoneal wall showed arteriolar endothelial cells expres-sing CD34 (Figure 2B) and several macrophages expressingCD68 (Figure 2C). Neither of the markers were expressedby peritoneal fibrocytes. Similar findings were observedon the peritoneal biopsy of Patient 2.

H&E-stained sections of the peritoneal tissue of bothcontrol patients showed a well-preserved peritoneal mem-brane (Figure 2D). Immunoperoxidase staining showed ar-teriolar endothelial cells expressing CD34 (Figure 2E) andnumerous macrophages expressing CD68 (Figure 2F).

Conventional electron microscopy and ESI

Electron microscopy analysis of Patients 1 and 2 peritonealtissues showed abundant collagen and scattered elastic fi-bres. There were also some electron-optically dark granu-lar deposits recognized as mast cell granules andartificially condensed chromatin. In both patients, very ti-ny (0.1–2.0 μm, mostly approximately 0.3 μm diameter)singular/multiple electron-optically dark inclusions werefound in cell protrusions (probable macrophages) dissem-inated in the connective tissue. The ESI and EELS spectralanalysis of those inclusions was negative for gadoliniumand calcium, but positive for iron in some of them (Figure3A–C). Peritoneal tissues from both controls only showedmoderate fibrotic tissue with blood vessels.

A

E

F

C

D

B

Fig. 2. Parietal peritoneal biopsies of Patient 1 with EPS (A–C) and Control 1 (D–F): (A) denuded mesothelium and a thickened, highly vascularizedsubmesothelial fibrous band, together with normal adjacent adipose tissue [the upper part of the submesothelial fibrous band has a hypocellularfibrohyalin appearance (H&E, magnification ×20)]; (B) immunoperoxidase staining of the peritoneal wall showing arteriolar endothelial cellsexpressing CD34 (magnification ×20); (C) immunoperoxidase staining of the peritoneal wall showing several macrophages expressing CD68(magnification ×40); (D) well-preserved peritoneal membrane (magnification ×20); (E) immunoperoxidase staining showing arteriolar endothelialcells expressing CD34 (magnification ×20); (F) immunoperoxidase staining showing numerous macrophages expressing CD68 (magnification ×20).

Absence of gadolinium deposits in patients with EPS 1331

Dow



ovember 2018

Discussion

The absence of clinical signs of NSF in both our EPS pa-tients (despite the exposure to linear chelates of gadolini-um, i.e. those with the highest risk of toxicity) [4,18],together with the peritoneal biopsy examinations thatshowed a highly f ibrotic process without CD34 andCD68 immunostaining in peritoneal fibrocytes or electronmicroscopic gadolinium deposits, argue against the impli-cation of gadolinium in the EPS development of both ourPD patients.

The diagnosis of NSF in the skin relies on histopatho-logic signs of fibrosis together with the presence in the der-ma of fibrocytes and histiocytic cells expressing CD34 (amarker of endothelial cells) and CD68 (a marker of macro-phages) antigens, respectively. We looked for the presenceof those cells within the fibrous peritoneal tissue of twoEPS patients in comparison with two control PD patientswithout signs of EPS. We found a similar strong expres-

sion of CD34 within the peritoneum of both EPS and con-trol patients, not by fibrocytes as it can be seen in NSFpatients, but by the numerous peritoneal capillaries. Thislatter observation reflects the neoangiogenesis processcommonly associated with long-term PD [39]. Likewise,a virtually not different strong expression of CD68 by nu-merous resident interstitial macrophages was found in bothEPS and control peritoneal membranes, which is a com-mon finding in the peritoneal membrane of long-termPD patients [40].

In patients with NSF, the pivotal role of gadolinium wassupported by the demonstration of the presence of gadolin-ium and of iron chelates within the derma using ESI andEELS analyses [14].

In addition, tissue fibrosis associated with the deposi-tion of gadolinium chelates has been demonstrated in nu-merous tissues, including the liver, the heart and thevessels [16,17]. In the present study, we could not detectany gadolinium chelates within the peritoneal membrane

A

C

B

1000,0

100,0

1140,0 1160,0 1180,0 1200,0Energy Loss

Energy Loss

Inte

nsity

Inte

nsity

1220,0 1240,0 eV

eV

Digits

Digits

40,0

20,0

0,0

650,0 670,0 690,0 710,0 730,0 750,0 770,0

Parallel EELS Gd - M5

Parallel EELS Fe - L3

Gd reference spectrum

Fe reference spectrum

recorded spectrum

recorded spectrum

Fig. 3. ESI and parallel EELS: (A) iron mapping on electron-optically dark inclusions found in cell protrusions (probable macrophages) disseminatedin the peritoneal connective tissue at high magnification (original magnification ×10 000), the net iron signal image is superimposed on the invertedHCI image showing the precise localization of the signal as a red spot on the tissue structure; (B) parallel EELS spectrum [the confirmation of aparticular element is demonstrated by its defined energy-loss edge at the energy level necessary for atom inner shell ionization; record of themeasured gadolinium signal (red) does not correspond to the reference gadolinium spectrum (green), allowing to exclude the presence ofgadolinium in the peritoneal tissue]; (C) parallel EELS spectrum [record of the measured iron signal (red) corresponds to the reference ironspectrum (green), allowing to document the presence of iron deposits in the peritoneal tissue].


Dow



ovember 2018

of both our EPS and control patients (despite numeroussamples and step sections examined). This latter point isprobably the key element to innocent gadolinium in the de-velopment of EPS. A word of caution has, however, to beraised since some iron-positive signals have been found inthe peritoneal tissues of both patients; a finding that hasalso been seen in association with gadolinium deposits[14,16]. A contamination of the present samples by iron-containing microtome blade can be excluded by the use ofa diamond knife and by strict contact avoidance betweenthe samples with iron-containing surfaces [14]. Though atransmetalation phenomenon cannot be entirely ruled out[18,41], iron load, due to multiple IV iron infusions to raisehaemoglobin level, as frequently observed in dialysis pa-tients, is more likely to explain this finding [42,43]. Also,the peritoneal biopsies have been taken in the parietal peri-toneum; we, however, do not think that the visceral perito-neum might have shown different results.

The lag time between NSF development and gadoliniuminjection is usually below 3months according to bothMarckmann [10] and Swaminathan [16]. In both patientswith EPS from the present study, the delay between gado-linium injection and EPS diagnosis was 9 and 20months,respectively. It seems thus unlikely that gadolinium couldhave triggered the fibrotic process of EPS and have subse-quently disappeared totally from the peritoneal tissues, giv-en its high tissular affinity and long half-life. We, however,have to acknowledge that the remoteness of the gadoliniumexposure in our patients and the limited peritoneal sam-plings represent the main limitations of the present study.Still, Schroeder et al. recently found persisting dermal gad-olinium deposits in NSF patients more than 3years afterthe last gadolinium administration [14].

Finally, the absence of gadolinium exposure in one pa-tient from our recent post-renal TP EPS series [6] furthersupports the absence of link between EPS and gadolinium.

Conclusion

The present observation implies that a search for anotheraetiopathogenic factor than gadolinium injection should bepursued in EPS following PD. The risk of EPS develop-ment after PD therapy is, therefore, not yet behind us withthe avoidance of gadolinium injection in PD patients.

Conflicts of interest statement. None declared.

References

1. Chin AI, Yeun JY. Encapsulating peritoneal sclerosis: an unpredict-able and devastating complication of peritoneal dialysis. Am J KidneyDis 2006; 47: 697–712

2. Summers AM, Clancy MJ, Syed F et al. Single-center experience ofencapsulating peritoneal sclerosis in patients on peritoneal dialysisfor end-stage renal failure. Kidney Int 2005; 68: 2381–2388

3. Balasubramaniam G, Brown EA, Davenport A et al. The Pan-Thames EPS study: treatment and outcomes of encapsulating perito-neal sclerosis. Nephrol Dial Transplant 2009; 24: 3209–3215

4. Perazella MA. Current status of gadolinium toxicity in patients withkidney disease. Clin J Am Soc Nephrol 2009; 4: 461–469

5. Fieren MWJA, Betjes MGH, Korte MR et al. Posttransplant encap-sulating peritoneal sclerosis: a worrying new trend? Perit Dial Int2007; 27: 619–624

6. Goffin E, Decleire PY. Does the incidence of encapsulating peritone-al sclerosis increase after renal transplantation? Perit Dial Int 2008;28: S5

7. Korte MR, Yo M, Betjes MGH et al. Increasing incidence of severeencapsulating peritoneal sclerosis after kidney transplantation. Ne-phrol Dial Transplant 2007; 22: 2412–2414

8. Cowper SE, Robin HS, Steinberg SM et al. Scleromyxoedema-likecutaneous diseases in renal-dialysis patients. Lancet 2000; 356:1000–1001

9. Grobner T. Gadolinium—a specific trigger for the development ofnephrogenic fibrosing dermopathy and nephrogenic systemic fibro-sis? Nephrol Dial Transplant 2006; 12: 3604–3605

10. Marckmann P, Skov L, Rossen K et al. Nephrogenic systemic fibrosis:suspected causative role of gadodiamide used for contrast-enhancedmagnetic resonance imaging. J Am Soc Nephrol 2006; 17: 2359–2362

11. Sadowski EA, Bennett LK, Chan MR et al. Nephrogenic systemicfibrosis: risk factors and incidence estimation. Radiology 2007;243: 148–157

12. High WA, Ayers RA, Chandler J et al. Cowper SE gadolinium is de-tectable within the tissue of patients with nephrogenic systemic fibro-sis. J Am Acad Dermatol 2007; 56: 21–26

13. High WA, Ayers RA, Cowper SE. Gadolinium is quantifiable withinthe tissue of patients with nephrogenic systemic fibrosis. J Am AcadDermatol 2007; 56: 710–712

14. Schroeder JA, Weingart C, Coras B et al. Ultrastructural evidence ofdermal gadolinium deposits in a patient with nephrogenic systemicfibrosis and end-stage renal disease. Clin J Am Soc Nephrol 2008;3: 968–975

15. Swaminathan S, Horn TD, Pellowski D et al. Nephrogenic systemicfibrosis, gadolinium, and iron mobilization. N Engl J Med 2007; 357:720–722

16. Swaminathan S, High WA, Ranville J et al. Cardiac and vascular met-al deposition with high mortality in nephrogenic systemic fibrosis.Kidney Int 2008; 73: 1413–1418

17. Kay J, Bazari H, Avery LL et al. Case records of the MassachusettsGeneral Hospital. Case 6-2008. A 46-year-old woman with renal fail-ure and stiffness of the joints and skin. N Engl J Med 2008; 358:827–838

18. Prchal D, Holmes DT, Levin A. Nephrogenic systemic fibrosis: thestory unfolds. Kidney Int 2008; 73: 1135–1137

19. Cowper SE, Su LD, Bhawan J et al. Nephrogenic fibrosing dermo-pathy. Am J Dermatopathol 2001; 23: 383–393

20. Lim YL, Lee HY, Low SC et al. Possible role of gadolinium in ne-phrogenic systemic fibrosis: report of two cases and review of theliterature. Clin Exp Dermatol 2007; 32: 353–358

21. Murashima M, Drott HR, Carlow D et al. Removal of gadolinium byperitoneal dialysis. Clin Nephrol 2008; 69: 368–372

22. Tsai CW, Chao CC, Wu VC et al. Nephrogenic fibrosing dermopathyin a peritoneal dialysis patient. Kidney Int 2007; 72: 1294

23. Othersen JB, Maize JC, Woolson RF et al. Nephrogenic systemic fi-brosis after exposure to gadolinium in patients with renal failure. Ne-phrol Dial Transplant 2007; 22: 3179–3185

24. Marckmann P, Skov L, Rossen K et al. Case–control study of gado-diamide-related nephrogenic systemic fibrosis. Nephrol Dial Trans-plant 2007; 22: 3174–3178

25. Reimer L, Deininger C, Egerton RF et al. Electron-Filtering Trans-mission Electron Microscopy. Berlin: Springer, 1995

26. Leapman RD, Sun SQ, Hunt JA et al. Biological electron energy lossspectroscopy in the field-emission scanning transmission electron mi-croscope. Scanning Microsc Suppl 1994; 8: 245–258 [discussion258–259]

27. Egerton RF. Electron Energy-Loss Spectroscopy in the Electron Mi-croscope. New York: Plenum, 1986

28. Simon GT, Heng YM. Electron energy loss spectroscopic imaging inbiology. Scanning Microsc 1988; 2: 257–266

29. Egerton RF. New techniques in electron energy-loss spectroscopy andenergy-filtered imaging. Micron 2003; 34: 127–139

Absence of gadolinium deposits in patients with EPS 1333

Dow



ovember 2018

30. Egerton RF. Limits to the spatial, energy and momentum resolutionof electron energy-loss spectroscopy. Ultramicroscopy 2007; 107:575–586

31. Keast VJ, Bosman M. New developments in electron energy lossspectroscopy. Microsc Res Tech 2007; 70: 211–219

32. Kapp N, Studer D, Gehr P et al. Electron energy-loss spectroscopy asa tool for elemental analysis in biological specimens. In: K John (ed).Electron Microscopy, Methods and Protocols. 2nd ed. Totowa: Hu-mana, 2007; 431–447

33. Kapp N, Kreyling W, Schulz H. Electron energy loss spectroscopy foranalysis of inhaled ultrafine particles in rat lungs. Microsc Res Tech2004; 63: 298–305

34. Rothen-Rutishauser BM, Schurch S, Haenni B et al. Interaction offine particles and nanoparticles with red blood cells visualized withadvanced microscopic techniques. Environ Sci Technol 2006; 40:4353–4359

35. Geiser M, Rothen-Rutishauser B, Kapp N et al. Ultrafine particlescross cellular membranes by nonphagocytic mechanisms in lungsand in cultured cells. Environ Health Perspect 2005; 113: 1555–1560

36. Hirai K, Pan J, Shimada H et al. Cytochemical energy-filteringtransmission electron microscopy of mitochondrial free radical forma-tion in paraquat cytotoxicity. J Electron Microsc (Tokyo) 1999; 48:289–296

37. Schlotzer-Schrehardt U, Kortje KH, Erb C. Energy-filtering transmis-sion electron microscopy (EFTEM) in the elemental analysis of pseu-doexfoliative material. Curr Eye Res 2001; 22: 154–162

38. Jonas L, Fulda G, Kroning G et al. Electron microscopic detection oftin accumulation in biliopancreatic concrements after induction ofchronic pancreatitis in rats by di-n-butyltin dichloride. UltrastructPathol 2002; 26: 89–98

39. Combet S, Miyata T, Moulin P et al. Vascular proliferation and en-hanced expression of endothelial nitric oxyde synthase in humanperitoneum exposed to long-term peritoneal dialysis. J Am Soc Ne-phrol 2000; 11: 717–728

40. Di Paolo N, Sacchi G. Atlas of peritoneal histology. Perit Dial Int2000; 20: S5–S96

41. Idee JM, Port M, Raynal I et al. Clinical and biological consequencesof transmetallation induced by contrast agents for magnetic resonanceimaging: a review. Fundam Clin Pharmacol 2006; 20: 563–576

42. Kovesdy CP. Iron and clinical outcomes in dialysis and non-dialysis-dependent chronic kidney disease patients. Adv Chronic Kidney Dis2009; 16: 109–116

43. Wish JB. Assessing iron status: beyond serum ferritin and transferrinsaturation. Clin J Am Soc Nephrol 2006; 1: S4–S8

Received for publication: 13.9.09; Accepted in revised form: 13.11.09


Dow



ovember 2018

absence of gadolinium deposits in the peritoneal membrane of

Documents