© 1990 S. KargcrAG, Basel 0250-8095/90/0l05-0389$2.7$/0
Am J Nephrol 1990;10:389-396
Systemic Involvement of Dialysis-Amyloidosis Josep M. Campistola, ManeI Soléb. José Munoz-Gomezc, José Lopez-Pedreta, Luis Reverta “Nephrology Service, bDepartment of Pathology, and ‘ Rheumatology Service, Hospital Clinic i Provincial. University of Barcelona, Spain
Key Words. Hemodialysis • Amyloidosis • P2 -Microglobulin • Visceral involvement • Two-dimensional echocardiogram
Introduction During the last 4 years, dialysis amyloidosis has emerged as an important and frequent complication in patients with chronic renal failure on maintenance he modialysis, especially after 7 or 8 years of substitution treatment [1-5]. The main clinical manifestations are development of a carpal tunnel syndrome, frequently bilateral and re lapsing, and amyloid arthropathy, characterized by per sistent swelling and effusions, mainly in the knees and shoulders, lytic bone lesions, destructive arthropathies, and pathological hip fractures [1-14]. Erosive spondy
loarthropathy is also common in this group of patients, although its relationship with amyloid deposits is contro versial [15-18]. In 1985 Gejyo ct al. [19] and Shirahama et al. [20] identified the plasmatic protein fc-microglobulin, with exclusively renal metabolism and with a sequence of amino acids similar to that of the constant domains of IgG as the major constituent protein of this new type of amyloidosis. An important question is whether the tissue distribu tion of dialysis amyloidosis is limited to osteoarticular structure or whether it may have a systemic and more extensive character. Although a few cases with asymp
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Abstract. A new type of amyloidosis, predominantly osteoarticular, has recently been recognized in uremic patients on hemodialysis, P2-microglobulin being the major constituent protein. Nowadays, it is not clear whether the amyloid deposition is limited to osteoarticular structures or whether it has a systemic character. In order to investigate the extension of dialysis amyloidosis, we studied 26 patients receiving hemodialysis treatment (mean time 12.2 years) for chronic renal failure due to nonamyloid nephropathy and who were affected by symptomatic dialysis amyloidosis. Twenty-two patients developed a carpal tunnel syndrome, and amyloid arthropathy was pres ent in 21. Subcutaneous abdominal fat aspiration, rectal and skin biopsy, and two-dimensional echocardiography were performed in most of the patients, searching for the visceral involvement. Surgical pieces (one stomach and two colon) and three necropsies of symptomatic patients were included in the systemic investigation. Also, we studied five necropsies of patients without articular symptoms. Histological confirmation of amyloid visceral involvement was demonstrated in 15 (58%) of the 26 patients studied. When positive two-dimensional echocardiograms were included, the percentage increased to 81 %. No differences in the rate of visceral involvement could be found between the two clinical groups (with and without carpal tunnel syndrome). Two-dimensional echocardiography represents the most useful tool to search for the visceral involvement of ^-microglobulin amyloidosis, followed by abdominal fat aspiration and rectal biopsy. Amyloid deposits were resistent to potassium permanganate treatment and reacted with antihuman fo-microglobulin (avidin-biotin-peroxidase method).
Campistol/Solé/Munoz-Gomez/Lopez-Pedret/Revert
tomatic visceral involvement have been reported in the literature [20-26], the systemic character of this new type of amyloidosis remains uncertain. We have studied a group of patients with chronic renal failure on hemodialysis and proven dialysis amy loidosis in order to investigate the systemic involvement of Pa-microglobulin amyloidosis. Patients and Methods Between 1981 and 1987, 412 patients on long-term hemodialysis have been treated at our institution, with a minimal dialysis period of 6 months. Most of the patients were dialyzed three times weekly during 4-5 h, with hollow-fiber cuprophane membrane without reuse. During the study period, 35 patients (9% of the total group) have developed a symptomatic dialysis amyloidosis with histological con firmation; 26 of the 35 amyloidotic patients were examined for vis ceral involvement. In all these patients at least two of the following examinations were performed: subcutaneous abdominal fat aspira tion, skin biopsy, two-dimensional echocardiography, and rectal biopsy. Surgical specimens (1 stomach and 2 colon) and three necropsies of these patients were studied. Also, we included five necropsy studies of dialyzed patients without symptomatic dialysis amyloidosis. The etiology of renal disease in the amyloidotic group was vari able: polycystic kidney disease in 7 patients, chronic glomerulone phritis in 4, nephrosclerosis in 1, uremic-hemolitic syndrome in 1, systemic lupus in I, and renal disease of unknown oriin in 6 patients. None of the patients had amyloidosis as primary renal dis ease. The mean age of the group studied was 60.6 ± 8.8 years (range 37-78 years), and the mean time on hemodialysis treatment was 12.2 ± 2.4 years (range 6-18 years). The clinical manfestations of the 26 patients (table 1) were amy loid carpal tunnel syndrome without amyloid arthropathy (n = 4) and carpal tunnel syndrome with amyloid arthropathy with the typ ical chronic oligoarthritis and demonstration of amyloid deposits in the synovial fluid sediment [27] or in the synovial membrane (n 22); 4 of them developed pathological fractures with massive amy loid deposits in the fractured bone. No statistical differences could be found between the two clinical groups (carpal tunnel syndrome with and without amyloid arthropathy) with regard to age and time on hemodialysis: for this reason, all 26 patients were considered as in the same group. None of the patients had clinical symptoms of visceral involvement. We also included in the study five necropsies of patients with chronic renal failure on hemodialysis and without clinical manifes tations of dialysis amyloidosis. The mean age of this asymptomatic patient group was 57.2 ± 17 years (range 33-77 years) without sig nificant differences with respect to the symptomatic group: the mean lime on hemodialysis was 5.2 ± 2.6 years (range 2-9 years) statistically different with respect to the symptomatic group (p < 0 . 0001 ) .
Diagnostic Exploration for Visceral Involvement Subcutaneous abdominal fa t aspiration represents for most groups the diagnostic procedure of choice in the evaluation of pos sible amyloid disease 28]. The specimen was obtained at bedside by
aspiration of subcutaneous abdominal fat with a 19-gauge needle and disposable syringe from two sterilized sub- or paraumbilical areas of the abdomen. Small pieces of the fat specimen were pressed between two clean glass slides, thus applying thin layers of fat tissue to each slide. Skin biopsy. Although its efficiency for the diagnosis of amyloid osis is low, Altemeyer et al. [29] have demonstrated amyloid sub stance in skin biopsies of 82% of chronic hemodialized patients treated for 8-13 years. For this reason we included skin biopsy in the examinations. A punch or excision biopsy of clinically unin volved skin, usually the forearm, was performed. Size and depth of the biopsy included the dermal blood vessels, sweat glands, skin appendages, and subcutaneous fat as well as dermis and epider mis. Two-dimensional échocardiographie examination for cardiac amyloidosis was first described by Siqueira-Filho et al. [30] and today represents a widely used tool to define cardiac involvement in amyloidotic diseases [31]. In our study, the echocardiogram was considered suggestive of amyloidosis only if an increased atrial sep tal thickness with increased myocardial echogenicity (‘granular spar kling’) was present. Rectal submucosa biopsy has been traditional in the diagnosis of amyloidosis [32]. In several patients a rectal biopsy was performed with the aim of obtaining submucosa because this tissue is involved more often than the mucosa in amyloid disease. Surgical specimens were also studied for amyloid infiltration in 1 patient undergoing gastrectomy for signet-ring cell carcinoma and 2 patients undergoing left hemicolectomy for diverticulitis. Necropsy examinations were performed in 8 patients: 3 with symptomatic (T-microglobulin amyloidosis and 5 without clinical manifestations of dialysis amyloidosis. Visceral tissues (lungs, heart, kidneys, bowels, liver, central nervous system, thyroid, etc.) and bone structures were exhaustively examined for amyloid infiltra tion. Pathology Studies Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Serial sections were obtained from paraffin blocks. Stains were performed with hematoxylin-eosin and alkaline Congo red with and without pretreatment with potassium perman ganate, according to the method of Wright et al. [33], Sections from skin biopsies were also stained with thioflavine T and crystal violet. Stained slides were investigated using conventional and polarized light microscopy. The remaining sections were designated to the immunohistochemical study. This was performed using the avidinbiotin-peroxidase technique [34], Rabbit antihuman Pa-microglobu lin antibody at a dilution of 1/600 and rabbit antihuman P compo nent antibody at a dilution of 1/1,600 were used as primary antisera. Secondary antiserum was biotinylated-swine antirabbit IgG. Con trol was performed by replacing the primary antiserum by phos phate-buffered saline. Positivity and specificity of the reactions were evaluated by comparing the sections tested with those stained with Congo red. Additional tissue samples corresponding to amyloidosis of dif ferent types were used as controls. These were from a case with medullary carcinoma of thyroid gland, from 2 autopsied cases with secondary amyloidosis, from carpal tissue of a case with primary amyloidosis, from carpal tissue of a case with myeloma-related amy loidosis, and from a case with nasal amyloid tumor associated with plasmacytoma.
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.
Table 1 Clinical characteristics of symptomatic patients and localization of amyloid deposits Patient No.
Age years
Sex
Nephropathy
Time on AA hemodialysis years
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
65 60 78 62 52 61 52 69 65 72 63 37 48 58 51 71 58 63 62 61 60 56 75 52 62 62
M M F F M M M M F F F F F M M F F M M F M F M M F F
chronic GN nephrosclerosis unknown PKD unknown chronic GN unknown nephrosclerosis unknown CIN U-H Sdr chronic GN Lupus nephrosclerosis reflux N PKD nephrosclerosis PKD PKD PKD unknown PKD nephrosclerosis chronic GN unknown PKD
13 11 10 11 13 13 14 13 13 11 13 17 15 12 9 11 12 14 11 10 11 12 6 18 11 13
+ + + + + + + + + + + + + + + + + + + + +
CTS
SAFA
bilateral bilateral bilateral bilateral bilateral bilateral bilateral bilateral bilateral right bilateral right bilateral bilateral right left bilateral left bilateral bilateral bilateral left
-
+ + + + + + + + +
SB
RB
-
+ + -
-
+ -
-
-
Others
+
stomach necropsy
+ + + + + + + + + +
-
Echoc
-
necropsy
PF colon
colon
+ -
-
PF PF necropsy
AA = Amyloid arthropathy; CTS = carpal tunnel syndrome; SAFA = subcutaneous abdominal fat aspiration; SB = skin biopsy; RB = rectal biopsy; Echoc - two-dimensional echocardiogram; M = male; F = female; GN = glomerulonephritis; PKD = polycystic kidney disease; CIN = chronic interstitial nephritis; U-H Sdr - uremic-hemolytic syndrome; Lupus = systemic Lupus erythematosus; Reflux N = backflow hydronephrosis; PF - pathological fracture.
In the whole group (n = 26) histological confirmation of amyloid visceral involvement could be demonstrated in 58% of the studied patients. When positive twodimensional echocardiograms were included, the rate with systemic involvement increased to 81 %. In the group of patients with carpal tunnel syndrome alone (n = 4), the rate with visceral involvement was 60% in both circumstances and in patients with amyloid arthropathy (n = 22) the rate of visceral involvement was 57% by histological examination and 81 % when includ ing positive two-dimensional echocardiograms. No sta tistical differences in the rate of visceral involvement could be found between the two groups (table 2).
Table 2. Frequency (%) of osteoarticular and visceral amyloid deposits in the two clinical groups Patients with articular symptoms
Osteoarticular Visceral Histologic Histologic and échocardiographie
CTS (n - 5)
AA (n - 21)
Patients without articular symptoms (5 necropsies)
100
100
20
60
57
0
60
81
-
CTS = Carpal tunnel syndrome; AA = Amyloid arthropathy.
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Results
Subcutaneous Abdominal Fat Aspiration Needle aspiration of the abdominal fat was per formed in 25 patients, being positive in 9 (36%) patients. Amyloid deposition was present in septa and vascular walls, but it was often scant and difficult to detect. Skin Biopsy All of the skin specimens (16 patients) were negative. No traces of amyloid could be observed in any of the cutaneous structures. Rectal Biopsy Rectal biopsy was performed in 12 patients. Positive results were obtained in 3 out of the 7 patients in whom the sample included submucosa (43%). In the other 5 negative samples, no submucosal tissue was obtained; therefore, amyloid infiltration could not be excluded. Two-Dimensional Echocardiogram Bidimensional echocardiography was performed in 23 patients. The échocardiographie examination of 12 (52%) out of the 23 patients studied was suggestive of cardiac amyloidosis, given the presence of nodular struc tures of the interventricular septum (‘granular spark ling') and increased atrial septum thickness. Surgical Specimens Stomach (n = 1). A massive and diffuse deposit of amyloid substance was found mainly in the external layer of muscularis propria (fig. 1). Also vascular subendothelial deposits were seen in the submucosa and in the muscular gastric wall.
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Sigmoid Cohn (n = 2). In both cases histological examination showed nodular subendothelial amyloid deposits in arterioles and small arteries of the intestinal submucosa (fig. 2), leading to a total or subtotal obstruc tion of the arteriolar lumen in some vessels. Focal depo sition in muscularis propria was observed in 1 case. Autopsies o f Amyloidotic Patients Patient No. 2. A 60-year-old man on hemodialysis during the last 11 years for chronic renal failure second ary to nephrosclerosis. Clinically bilateral amyloid car pal tunnel syndrome was observed. He died from a lung carcinoma. Necropsy showed nodular amyloid deposits in the subendothelium of arterioles and small arteries of intestinal submucosa, heart, liver, lungs, testicles, and adrenal glands. Moreover, diffuse deposits were found in heart, prostate, and perithyroid tissue. No amyloid de posits were found in kidney, pancreas, spleen, and cen tral nervous system. Patient No. 10. A 72-year-old women on maintenance hemodialysis since 1977 because of a chronic interstitial nephritis. In 1983 she underwent release of a right carpal tunnel syndrome, demonstrating amyloid substance in the surgical specimen. In 1984. she complained of pain ful persistent effusion and swelling in knees and should ers, and amyloid origin was confirmed by a synovial biopsy. In 1986 the patient was admitted because of a pathological fracture of the left femur neck. Massive amyloid infiltration was observed in the fractured bone. A biopsy of the subcutaneous abdominal fat was per formed and proved to be amyloid positive. A twodimensional echocardiogram showed nodular images in
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Fig. 2. Subcndothclial amyloid deposit in the arteriolar wall of rectal submucosa, reacting strongly with fL-micoglobulin antiserum. Avidin-biotin-peroxidase complex. X 300.
the interventricular septum, suggestive of cardiac amy loidosis (fig. 3a). Rectal and skin biopsies discarded the presence of amyloid substance. The patient died in April 1988 secondary to an aluminum encephalopathy. Nec ropsy discarded visceral involvement, except for the presence of nodular amyloid deposits (3-5 mm in diam eter) in the myocardium (fig. 3b). Immunohistochemical examination confirmed the (^-microglobulin origin of the myocardial amyloid deposits. Patient No. 26. A 62-year-old woman affected by chronic renal failure secondary to polycystic kidney dis ease and on maintenance hemodialysis during the last 13 years. Two years prior to death, she underwent surgery for a left carpal tunnel syndrome, and amyloid substance was found in the transverse carpal ligament. One year later, she complained of right-knee pain, with swelling and effusion, and amyloid substance was found in the synovial fluid sediment. Subcutaneous abdominal fat aspiration was positive for amyloid deposits. Two-di mensional echocardiography excluded cardiac involve ment. The patient died from a Legionella pneumonia. Necropsy showed nodular amyloid deposits in the sub endothelium of arterioles and small arteries of the intes tinal submucosa. Also amyloid deposits were confirmed in the osteoarticular structures. The rest of the visceral specimens were negative for Congo red stain.
massive amyloid deposits in osteoarticular structures, with severe involvement of synovial membrane and cap sules of shoulders and knees. No amyloid substance was demonstrated in visceral tissues. Necropsy was negative (visceral and bone tissues) for amyloid deposition in the remaining 4 asymptomatic patients. The rate of visceral involvement was statisti cally highly sinificant in the symptomatic patient group (p < 0.02) and clearly related to the time on hemodialy sis (p < 0.0001; table 2).
Autopsies o f Asymptomatic Patients A 72-year-old man with nephrosclerosis and on main tenance hemodialysis during the last 4 years. He had no related clinical manifestations of dialysis amyloidosis. He died from myocardial infarction. Necropsy showed
Pathology Studies Amyloid substance exhibited the characteristic stain with Congo red. Following treatment with potassium permanganate a partial loss of congophilia was observed, but green birefringence was clearly maintained.
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Fig. 3. a Left: two-dimensional echocardiogram showing nodular structures at the interventricular septum ('granular sparkling'; ar rows), suggestive of cardiac amyloidosis. VD = Right ventricule; VI = left ventricule; AD = right auricule; AI - left auricule. b Right: nodular deposition of amyloid substance in the myocardium. Congo red. X 20.
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(L-Microglobulin was positive in all tested tissues. Areas with positive reactions corresponded well with Congo red positive deposits in parallel sections. All the specimens used as controls were negative. Amyloid P component was positive in nearly all tested samples, including cardiac and intestinal deposits.
Discussion Dialysis amyloidosis has recently been recognized as a new complication in the osteoarticular area in long term hemodialyzed patients. The first description of this new type of amyloidosis was in 1980, when Assenat et al. [35] demonstrated the amyloid origin of the carpal tun nel syndrome in hemodialyzed patients. Afterward, in 1984, Charra et al. [36] reported the high association of amyloid carpal tunnel syndrome with shoulder pain in patients on hemodialysis. Dialysis amyloidosis involves predominantly osteoarticular structures, and it has been found most often to be associated with destructive ar thropathy, destructive spondyloarthropathy, pathologi cal fractures, and carpal tunnel syndrome [1-14], In par ticular, it has been detected in synovium, capsula, sub chondral bone, and in intervertebral disks. Gejyo et al. [19] and Shirahama et al. [20] in 1985 identified the p2 microglobulin as the major constituent protein of this new type of amyloidosis. The question whether such amyloidosis is localized to wrists and contiguous structures, confined to the muscu loskeletal system, or widely disseminated has remained controversial. The extent of organ involvement due to amyloid may have prognostic significance for these pa tients. At present, marked extra-articular deposits have been only reported sporadically. However, some faint p2 microglobulin amyloid deposition has been demon strated in the wall of arterioles of several visceras, including heart, lungs, liver, and intestine [20, 21, 2326, 37-39], In all these reported patients, the visceral involvement was asymptomatic except in 3 cases re ported by Fuchs et al. [22] who developed gastrointesti nal bleeding associated with vascular amyloid deposits in one and odynophagia associated with lingual amyloid infiltration in the other and in the case reported by Shinoda et al. [39] with intestinal pseudo-obstruction. Systemic studies have failed to show amyloid deposits in abdominal fat aspiration and in skin biopsy [12. 26, 32]. Recently, Varga et al. [40] have reported the lack of amyloid in abdominal fat aspirates from 30 patients undergoing long-term hemodialysis, although only 11
patients complained of carpal tunnel syndrome and nei ther of them with histological confirmation of P2-microglobulin amyloid origin. Altemeyer et al. [29] in 1983 suspected a high incidence of skin amyloid deposits in hemodialyzed patients, but revised their view later, stat ing that the Congo red positive material detected in the corium of the skin of dialized patients was most proba bly altered collagen [41], Rectal biopsy specimens in patients affected by dialysis amyloidosis have shown small perivascular amyloid deposits on the submucosa layer in 6 of 9 reported patients, but none of these patients had clinically significant amyloid involvement [2, 20, 25]. Liver biopsy performed once in a dialyzed patient with a P2-microglobulin amyloidosis was amy loid negative [6], Hawkins et al. [42], using l23I-labelled purified human serum amyloid P component, failed to find visceral deposits in 2 hemodialysis patients affected by P2 -microglobulin amyloidosis. We have previously reported visceral involvement in 3 hemodialyzed patients with amyloid deposits in the wall of the vessels of several viscera and demonstrated the P2 -microglobulin origin [21]. We then started a pro spective study to confirm the systemic character of dial ysis amyloidosis. The study, included 26 of our 35 patients with dialysis amyloidosis, in whom at least two systemic examinations were performed. Also, five nec ropsies of dialyzed patients without symptomatic dialy sis amyloidosis were included. In 15 of 26 patients (58%) amyloid deposits were histologically demonstrated in visceral structures and in 21 of these patients (81 %), if the positive two-dimensional echocardiograms were considered. Whereas the five necropsies of asymptom atic patients failed to demonstrate visceral involvement, and although none of the patients had had articular symptoms, one of them showed osteoarticular infiltra tion. None of the patients with visceral deposits had sys temic clinical manifestations. This higher incidence of visceral involvement in our patients could be due to sev eral factors: the long time on hemodialysis (mean 12 years), the advanced age (mean 60 years), and the selected group of studied patients, all of them affected by symptomatic dialysis amyloidosis. Abdominal fat aspiration represents for most groups the best diagnostic procedure in the evaluation of sys temic amyloid disease [28]. Up to now. all abdominal fat samples of dialyzed patients were negative for (^-microglobulin amyloid, including our first description of amy loid arthropathy in long-term hemodialysis patients in which six abdominal fat specimens were amyloid nega tive [1, 23, 38]. In the present study, amyloid was present
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Campistol/Sole/Munoz-Gomez/Lopez-Pedret/Revert
Systemic Involvement of Dialysis-Amyloidosis
involvement (carpal tunnel syndrome and amyloid ar thropathy) and is followed by the visceral one with pro gressive nodular subendothelial amyloid infiltration. In future, with the increased age of the dialyzed population, visceral involvement would be more usual and presum ably with systemic symptoms. Acknowledgments This work was supported in part by grant CIRIT-AR87 and has been presented as a free communication at the Xth International Congress of Nephrology, London. England, July 1987. and at the XXIVth Congress of the EDTA-Europcan Renal Association, Berlin (West). October 1987.
References 1 Muñoz-Gómez J, Bergada E, Gómez R. Llopart E, Subías E, Rotes J, Solé M: Amyloid arthropathy in patients undergoing periodical haemodialysis for chronic renal failure: A new com plication. Ann Rheum Dis 1985;44:729-733. 2 Morita T. Suzuki M. Kamimura A, Hirasawa Y: Amyloidosis of a possible new type in patients receiving long-term hemodialysis. Arch Pathol Lab Med 1985;109:1029-1032. 3 Bardin T, Kuntz D. Zingraff J. Voisin MC. Zelmar A. Lansaman J: Synovial amyloidosis in patients undergoing long-term hemo dialysis. Arthritis Rheum 1985:28:1052—1058. 4 Brown EA. Arnold JR, Gower PE: Dialysis arthropathy: Compli cation of long-term treatment with hemodialysis. Br Med J 1986; 292:163-166. 5 Hardouin P, Flipo RM. Foissac-Gegoux P, Thevcnon A. Pouyol F. Duquesnoy B. Delcambre B: Current aspects of osteoarticular pathology in patients undergoing hemodialysis: Study of 80 patients. I. Clinical and radiological analysis. J Rheumatol 1987: 14:780-783. 6 Fenvcs A. Emctt M. White M. Greenway G. Michaels D: Carpal tunnel syndrome with cystic bone lesions secondary' to amyloid osis in chronic hemodialysis patients. Am J Kidney Dis 1986;7: 130-134. 7 Bergada E. Montoliu J. Subias R. Sole M. Lopez-Pedret J, Revert LL: Síndrome del túnel carpiano con deposito local y articular de sustancia amiloide en el hemodializado. Med Clin (Bare) 1986:86:319-322. 8 Huaux JP. Noël H. Bastien P. Malghem J. Maldaguc B. Dcvogelaer JP. Nagant de Deuxchaisnes C: Amvlose articulaire, fracture du col fémoral et hémodialyse périodique chronique. Rev Rhum Mal üstéoartic 1985:52:179-182. 9 Muñoz-Gómez J. Gómez R. Llopart E. Solé M: The clinical pic ture of amyloid arthropathy in patients with chronic renal fail ure maintained on hemodialysis using cellulose membranes. Ann Rheum Dis 1987:46:573-579. 10 Hadjipavlou A. Lander P, Beg:n L. Bercovitch D. Davidman M. Jakab E: Skeletal amyloidosis due to beta microglobulinemia in a patient on hemodialysis. J Bone Joint Surg 1988:70:119-121. 11 Schwarz A. Keller F. Scyfcrt S. Poll W, Molzahn M. Distler A: Carpal tunnel syndrome: A major complication in long-term hemodialysis patients. Clin Nephrol 1984;22:133-137.
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in septa and vascular wall of 9 out of the 26 abdominal fat specimens (36%); 3 of them were previously negative. Two-dimensional echocardiography represents a useful tool to define cardiac involvement in amyloid diseases, if increased atrial septal thickness with increased myo cardial echogenicity (‘granular sparkling’) is present [31], In our study, two-dimensional echocardiography was the most sensitive method to define visceral involvement and was suggestive of cardiac amyloidosis in 12 out of 23 patients studied (52%). Unfortunately, endocardium biopsy is too aggressive to practice routinely, but we have confirmed the presence of a positive echocardio gram and cardiac involvement in the necropsy of 1 patient (No. 26) who presented nodular amyloid depos its in the myocardium. There are no previous reports of two-dimensional echocardiography in p2-microglobulin amyloidotic patients, and we consider that more experi ence would be necessary to confirm our findings. Skin biopsy was negative for amyloid in all 16 speci mens, so we think that it is not a useful tool in this type of amyloidosis. Rectal biopsy was performed in 12 pa tients and demonstrated the presence of nodular suben dothelium amyloid deposits in arterioles and small arter ies of the rectal submucosa in three specimens. Amyloid infiltration was only shown in submucosa vessels; there fore, to investigate dialysis amyloidosis, rectal biopsy must be deep enough to contain rectal submucosa. In the surgical specimens (1 stomach and 2 colon) amyloid substance was demonstrated in all examina tions. Amyloid deposits fitted in two different patterns: in a diffuse form infiltrating the submucosa layer and in nodular subendothelial deposits in the vascular wall of arterioles and small arteries. In the three autopsies of dialysis amyloidosis patients visceral involvement was clearly demonstrated in all, with nodular subendothelial amyloid deposits in arteri oles of several organs and nodular deposits in endocar dium and myocardium. Amyloid substance reacted strongly with anti-p2-microglobulin and not with the other antisera, confirming the exclusively p2-microglobulin origin. Amyloid infiltration was demonstrated only in 1 of the 5 autopsies of the asymptomatic patients and only in osteoarticular structures and not in viscera. In summary, we think that dialysis amyloidosis has a systemic character with visceral involvement. Two-di mensional ehocardiography represents the most useful tool to search for the visceral involvement of p2-microglobulin amyloidosis, followed by abdominal fat aspira tion and rectal biopsy. The natural course of this new type of amyloidosis goes first with the osteoarticular
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12 DiRaimondo CR. Casey TT, DiRaimondo CV, Stone WJ: Pathologic fractures associated with idiopathic amyloidosis of bone in chronic hemodialysis patients. Nephron 1986;43:22— 27. 13 Csey TT. Stone WJ, DiRaimondo CR. Page DL, Gorevic PD: Dialysis-related amyloid is amyloid of beta-2-microglobulin (AM b2m) origin. Arthritis Rheum 1986;29:1170. 14 Zingraff J, Bardin T, Kuntz D, Voisin MC, Juquel JP, Driieke T: Degenerative osteo-articular lesions and amyloid infiltration in long-term hemodialysis patients. Proc Eur Dial Transplant As soc Eur Renal Assoc 1985;22:131-135. 15 Allain TJ, Stevens PE, Bridges LR. Phillips ME: Dialysis my elopathy: Quadriparesis due to extradural amyloid of (L-microglobulin origin. Br Med J 1988;296:752-753. 16 Kuntz D, Naveau B, Bardin T, Driieke T, Treves R, Dryll A: Destructive spondyloarthropathy in hemodialyzed patients: A new syndrome. Arthritis Reum 1984;27:369-375. 17 Muñoz Gomez J, Estrada P: Early radiologic manifestations of destructive spondylarthropathy in hemodialyzed patients. Ar thritis Reum 1986;29:1171-1172. 18 Sebert JL, Fardellone P, Marie A, Deramond H, Lambrey G, Legars D, Galibert P, Smajda A, Fournier A: Destructive spondylarthopathy in hemodialyzed patients. Possible role of amy loidosis. Arthritis Rheum 1986;29:301-303. 19 Gejyo F, Yamada T, Odani S, Nakagawa Y, Arakawa M, Kunitomo T, Kataoka H, Susuki M, Hirasawa Y, Shirahama T, Cohen AS, Schmid K: A new of amyloid protein associated with chronic hemodialysis was identified as (ii-microglobulin. Biochem Biophys Res Commun 1985;129:701-706. 20 Shirahama T, Skinner M, Cohen AS, Gejyo F, Arakawa M, Suzuki M, Hirasawa Y: Histochemical and immunohistochemi cal characterization of amyloid associated with chronic hemodi alysis as (îi-microglobulin. Lab Invest 1985;53:705-709. 21 Campistol JM, Cases A, Torras A, Muñoz-Gómez J, Solé M. Montoliu J, Lopez-Pedret J, Revert LL: Visceral involvement of dialysis amyloidosis. Am J Nephrol 1987;7:390-393. 22 Fuchs A, Jagirdar J, Schwartz IS: Beta-2-microglobulin amyloid osis (AB2M) in patients undergoing long-term hemodialysis. Am J Clin Pathol 1987;88:302-307. 23 Noël LH, Zingraff J, Bardin T, Atienza C, Kuntz D Driieke T: Tissue distribution of dialysis amyloidosis. Clin Nephrol 1987; 27:175-178. 24 Ogawa H, Saito A, Hirabayashi N, Hara K: Amyloid deposition in systemic organs in long-term hemodialysis patients. Clin Nephrol 1987;28:199-204. 25 Hardouin P, Flipo RM, Lecomte-Houcke M, Foissac-Gcgoux P, Delcambre B: Amylose des hémodialyses. Recherche d’une dif fusion générale par biopsie rectale. Presse Méd 1987; 16:445446. 26 Theaker JM. Raine AEG, Rainey AJ, Heryet A, Clark A, Oliver DO: Systemic amyloidosis of P;-microglobulin type: A complica tion of long-term hemodialysis. J Clin Pathol 1987;40:1247— 1251. 27 Muñoz-Gómez J, Gomez Perez R, Sole M, Llopart E: Synovial fluid examination for the diagnosis of synovial amyloidosis in patients with chronic renal failure undergoing hemodialysis. Ann Rheum Dis 1987;46:324-326. 28 Duston MA, Skinner M. Shirahama T, Cohen AS: Diagnosis of amyloidosis by abdominal fat aspiration. Analysis of four years’ experience. Am J Med 1987;82:412-414.
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29 Altemeyer P. Kachel HG, Runne U: Microangiopathy, altera tions of connective tissue, and deposition of an amyloid-like substance in patients with chronic renal failure. Hautarzt 1983; 34:277-285. 30 Siqueira-Filho AG, Cunha CLP, Tajik AJ, Seward JB, Schatten berg TT, Giuliani ER: M-mode and two-dimensional échocar diographie features in cardiac amyloidosis. Circulation 1981 ;63: 188-196. 31 Falk R. Plchn J, Deering T. Schick EC, Boinay P, Rubinow A, Skinner M, Cohen AS: Sensitivity and specificity of échocardio graphie features of cardiac ayloidosis. Am J Cardiol 1987;59: 418-422. 32 Janssen S, Van Rijswijk MH, Meijer S, Ruinen L, Van der Hem GK: Clinical evaluation of amyloid arthropathy and AL amyloid disease; in Marrink J, Van Rijswijk MH (eds): Amyloidosis. Dordrecht, Nijhoff, 1986, pp61-72. 33 Wright JR, Calkins E, Humphrey RL: Potassium permanganate reaction in amyloidosis. A histologic method to assist in differ entiating forms of this disease. Lab Invest 1977;36:274-281. 34 Hsu SM, Raine L, Fänger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelcd antibody (UAP) procedures. J Histochem Cytochem 1981;29:577-580. 35 Assenât U, Calemard E, Charra B, Laurent G, Terrât JC, Vanel T: Hémodialyse syndrome du canal carpien et substance amy loïde. Nouv Presse Méd 1980;9:1715. 36 Charra B, Calemard E, Uzan M, Terrât JC, Vanel T, Laurent G: Carpal tunnel syndrome, shoulder pain and amyloid deposits in long-term hemodialysis patients. Proc Eur Dial Transplant As soc Eur Renal Assoc 194;21:291-295. 37 Takahashi S, Morita T, Koda Y, Murayama H, Hirasawa Y: Gastrointestinal involvement of dialysis-related amyloidosis. Clin Nephrol 1988;30:168-171. 38 Floege J. Brandis A, Nonnast-Daniel B. Westhoff-Bleck M, Tiedow G, Linke RP, Koch KM: Subcutaneous amyloid tumor of beta-2-microglobulin origin in a long-term hemodialysis patient. Nephron 1989;53:73-75. 39 Shinoda T, Komatsu M, Aizawa T, Shirota T, Yamada T, Ehara T, Mizukami E: Intestinal pseudo-obstruction due to dialysis amyloidosis. Clin Nephrol 1989;32:284-289. 40 Varga J, Idelson BA, Felson D, Skinner M, Cohen AS: Lack of amyloid in abdominal fat aspirates from patients undergoing long-term hemodialysis. Arch Intern Med 1987; 147:14551457. 41 Kachel HG, Altemeyer P, Kühn KW, Koch KM. Baldamus CA: Deposition of non-amyloid material in connective tissue in urae mia. Blood Purif 1984;2:142-144. 42 Hawkins PN, Myers MJ, Lavender JP, Pepys MB: Diagnostic radionuclide imaging of amyloid: Biological targeting by circu lating human serum amyloid P component. Lancet 1988;i: 1413— 1418. Received: December 8, 1989 Accepted: April 11,1990 Dr. J.M. Campistol Nephrology Service Hospital Clinic i Provincial 170, Villarroel E-08036 Barcelona (Spain)
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Am J Nephrol 1990;10:389-396
Systemic Involvement of Dialysis-Amyloidosis Josep M. Campistola, ManeI Soléb. José Munoz-Gomezc, José Lopez-Pedreta, Luis Reverta “Nephrology Service, bDepartment of Pathology, and ‘ Rheumatology Service, Hospital Clinic i Provincial. University of Barcelona, Spain
Key Words. Hemodialysis • Amyloidosis • P2 -Microglobulin • Visceral involvement • Two-dimensional echocardiogram
Introduction During the last 4 years, dialysis amyloidosis has emerged as an important and frequent complication in patients with chronic renal failure on maintenance he modialysis, especially after 7 or 8 years of substitution treatment [1-5]. The main clinical manifestations are development of a carpal tunnel syndrome, frequently bilateral and re lapsing, and amyloid arthropathy, characterized by per sistent swelling and effusions, mainly in the knees and shoulders, lytic bone lesions, destructive arthropathies, and pathological hip fractures [1-14]. Erosive spondy
loarthropathy is also common in this group of patients, although its relationship with amyloid deposits is contro versial [15-18]. In 1985 Gejyo ct al. [19] and Shirahama et al. [20] identified the plasmatic protein fc-microglobulin, with exclusively renal metabolism and with a sequence of amino acids similar to that of the constant domains of IgG as the major constituent protein of this new type of amyloidosis. An important question is whether the tissue distribu tion of dialysis amyloidosis is limited to osteoarticular structure or whether it may have a systemic and more extensive character. Although a few cases with asymp
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Abstract. A new type of amyloidosis, predominantly osteoarticular, has recently been recognized in uremic patients on hemodialysis, P2-microglobulin being the major constituent protein. Nowadays, it is not clear whether the amyloid deposition is limited to osteoarticular structures or whether it has a systemic character. In order to investigate the extension of dialysis amyloidosis, we studied 26 patients receiving hemodialysis treatment (mean time 12.2 years) for chronic renal failure due to nonamyloid nephropathy and who were affected by symptomatic dialysis amyloidosis. Twenty-two patients developed a carpal tunnel syndrome, and amyloid arthropathy was pres ent in 21. Subcutaneous abdominal fat aspiration, rectal and skin biopsy, and two-dimensional echocardiography were performed in most of the patients, searching for the visceral involvement. Surgical pieces (one stomach and two colon) and three necropsies of symptomatic patients were included in the systemic investigation. Also, we studied five necropsies of patients without articular symptoms. Histological confirmation of amyloid visceral involvement was demonstrated in 15 (58%) of the 26 patients studied. When positive two-dimensional echocardiograms were included, the percentage increased to 81 %. No differences in the rate of visceral involvement could be found between the two clinical groups (with and without carpal tunnel syndrome). Two-dimensional echocardiography represents the most useful tool to search for the visceral involvement of ^-microglobulin amyloidosis, followed by abdominal fat aspiration and rectal biopsy. Amyloid deposits were resistent to potassium permanganate treatment and reacted with antihuman fo-microglobulin (avidin-biotin-peroxidase method).
Campistol/Solé/Munoz-Gomez/Lopez-Pedret/Revert
tomatic visceral involvement have been reported in the literature [20-26], the systemic character of this new type of amyloidosis remains uncertain. We have studied a group of patients with chronic renal failure on hemodialysis and proven dialysis amy loidosis in order to investigate the systemic involvement of Pa-microglobulin amyloidosis. Patients and Methods Between 1981 and 1987, 412 patients on long-term hemodialysis have been treated at our institution, with a minimal dialysis period of 6 months. Most of the patients were dialyzed three times weekly during 4-5 h, with hollow-fiber cuprophane membrane without reuse. During the study period, 35 patients (9% of the total group) have developed a symptomatic dialysis amyloidosis with histological con firmation; 26 of the 35 amyloidotic patients were examined for vis ceral involvement. In all these patients at least two of the following examinations were performed: subcutaneous abdominal fat aspira tion, skin biopsy, two-dimensional echocardiography, and rectal biopsy. Surgical specimens (1 stomach and 2 colon) and three necropsies of these patients were studied. Also, we included five necropsy studies of dialyzed patients without symptomatic dialysis amyloidosis. The etiology of renal disease in the amyloidotic group was vari able: polycystic kidney disease in 7 patients, chronic glomerulone phritis in 4, nephrosclerosis in 1, uremic-hemolitic syndrome in 1, systemic lupus in I, and renal disease of unknown oriin in 6 patients. None of the patients had amyloidosis as primary renal dis ease. The mean age of the group studied was 60.6 ± 8.8 years (range 37-78 years), and the mean time on hemodialysis treatment was 12.2 ± 2.4 years (range 6-18 years). The clinical manfestations of the 26 patients (table 1) were amy loid carpal tunnel syndrome without amyloid arthropathy (n = 4) and carpal tunnel syndrome with amyloid arthropathy with the typ ical chronic oligoarthritis and demonstration of amyloid deposits in the synovial fluid sediment [27] or in the synovial membrane (n 22); 4 of them developed pathological fractures with massive amy loid deposits in the fractured bone. No statistical differences could be found between the two clinical groups (carpal tunnel syndrome with and without amyloid arthropathy) with regard to age and time on hemodialysis: for this reason, all 26 patients were considered as in the same group. None of the patients had clinical symptoms of visceral involvement. We also included in the study five necropsies of patients with chronic renal failure on hemodialysis and without clinical manifes tations of dialysis amyloidosis. The mean age of this asymptomatic patient group was 57.2 ± 17 years (range 33-77 years) without sig nificant differences with respect to the symptomatic group: the mean lime on hemodialysis was 5.2 ± 2.6 years (range 2-9 years) statistically different with respect to the symptomatic group (p < 0 . 0001 ) .
Diagnostic Exploration for Visceral Involvement Subcutaneous abdominal fa t aspiration represents for most groups the diagnostic procedure of choice in the evaluation of pos sible amyloid disease 28]. The specimen was obtained at bedside by
aspiration of subcutaneous abdominal fat with a 19-gauge needle and disposable syringe from two sterilized sub- or paraumbilical areas of the abdomen. Small pieces of the fat specimen were pressed between two clean glass slides, thus applying thin layers of fat tissue to each slide. Skin biopsy. Although its efficiency for the diagnosis of amyloid osis is low, Altemeyer et al. [29] have demonstrated amyloid sub stance in skin biopsies of 82% of chronic hemodialized patients treated for 8-13 years. For this reason we included skin biopsy in the examinations. A punch or excision biopsy of clinically unin volved skin, usually the forearm, was performed. Size and depth of the biopsy included the dermal blood vessels, sweat glands, skin appendages, and subcutaneous fat as well as dermis and epider mis. Two-dimensional échocardiographie examination for cardiac amyloidosis was first described by Siqueira-Filho et al. [30] and today represents a widely used tool to define cardiac involvement in amyloidotic diseases [31]. In our study, the echocardiogram was considered suggestive of amyloidosis only if an increased atrial sep tal thickness with increased myocardial echogenicity (‘granular spar kling’) was present. Rectal submucosa biopsy has been traditional in the diagnosis of amyloidosis [32]. In several patients a rectal biopsy was performed with the aim of obtaining submucosa because this tissue is involved more often than the mucosa in amyloid disease. Surgical specimens were also studied for amyloid infiltration in 1 patient undergoing gastrectomy for signet-ring cell carcinoma and 2 patients undergoing left hemicolectomy for diverticulitis. Necropsy examinations were performed in 8 patients: 3 with symptomatic (T-microglobulin amyloidosis and 5 without clinical manifestations of dialysis amyloidosis. Visceral tissues (lungs, heart, kidneys, bowels, liver, central nervous system, thyroid, etc.) and bone structures were exhaustively examined for amyloid infiltra tion. Pathology Studies Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Serial sections were obtained from paraffin blocks. Stains were performed with hematoxylin-eosin and alkaline Congo red with and without pretreatment with potassium perman ganate, according to the method of Wright et al. [33], Sections from skin biopsies were also stained with thioflavine T and crystal violet. Stained slides were investigated using conventional and polarized light microscopy. The remaining sections were designated to the immunohistochemical study. This was performed using the avidinbiotin-peroxidase technique [34], Rabbit antihuman Pa-microglobu lin antibody at a dilution of 1/600 and rabbit antihuman P compo nent antibody at a dilution of 1/1,600 were used as primary antisera. Secondary antiserum was biotinylated-swine antirabbit IgG. Con trol was performed by replacing the primary antiserum by phos phate-buffered saline. Positivity and specificity of the reactions were evaluated by comparing the sections tested with those stained with Congo red. Additional tissue samples corresponding to amyloidosis of dif ferent types were used as controls. These were from a case with medullary carcinoma of thyroid gland, from 2 autopsied cases with secondary amyloidosis, from carpal tissue of a case with primary amyloidosis, from carpal tissue of a case with myeloma-related amy loidosis, and from a case with nasal amyloid tumor associated with plasmacytoma.
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.
Table 1 Clinical characteristics of symptomatic patients and localization of amyloid deposits Patient No.
Age years
Sex
Nephropathy
Time on AA hemodialysis years
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
65 60 78 62 52 61 52 69 65 72 63 37 48 58 51 71 58 63 62 61 60 56 75 52 62 62
M M F F M M M M F F F F F M M F F M M F M F M M F F
chronic GN nephrosclerosis unknown PKD unknown chronic GN unknown nephrosclerosis unknown CIN U-H Sdr chronic GN Lupus nephrosclerosis reflux N PKD nephrosclerosis PKD PKD PKD unknown PKD nephrosclerosis chronic GN unknown PKD
13 11 10 11 13 13 14 13 13 11 13 17 15 12 9 11 12 14 11 10 11 12 6 18 11 13
+ + + + + + + + + + + + + + + + + + + + +
CTS
SAFA
bilateral bilateral bilateral bilateral bilateral bilateral bilateral bilateral bilateral right bilateral right bilateral bilateral right left bilateral left bilateral bilateral bilateral left
-
+ + + + + + + + +
SB
RB
-
+ + -
-
+ -
-
-
Others
+
stomach necropsy
+ + + + + + + + + +
-
Echoc
-
necropsy
PF colon
colon
+ -
-
PF PF necropsy
AA = Amyloid arthropathy; CTS = carpal tunnel syndrome; SAFA = subcutaneous abdominal fat aspiration; SB = skin biopsy; RB = rectal biopsy; Echoc - two-dimensional echocardiogram; M = male; F = female; GN = glomerulonephritis; PKD = polycystic kidney disease; CIN = chronic interstitial nephritis; U-H Sdr - uremic-hemolytic syndrome; Lupus = systemic Lupus erythematosus; Reflux N = backflow hydronephrosis; PF - pathological fracture.
In the whole group (n = 26) histological confirmation of amyloid visceral involvement could be demonstrated in 58% of the studied patients. When positive twodimensional echocardiograms were included, the rate with systemic involvement increased to 81 %. In the group of patients with carpal tunnel syndrome alone (n = 4), the rate with visceral involvement was 60% in both circumstances and in patients with amyloid arthropathy (n = 22) the rate of visceral involvement was 57% by histological examination and 81 % when includ ing positive two-dimensional echocardiograms. No sta tistical differences in the rate of visceral involvement could be found between the two groups (table 2).
Table 2. Frequency (%) of osteoarticular and visceral amyloid deposits in the two clinical groups Patients with articular symptoms
Osteoarticular Visceral Histologic Histologic and échocardiographie
CTS (n - 5)
AA (n - 21)
Patients without articular symptoms (5 necropsies)
100
100
20
60
57
0
60
81
-
CTS = Carpal tunnel syndrome; AA = Amyloid arthropathy.
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Results
Subcutaneous Abdominal Fat Aspiration Needle aspiration of the abdominal fat was per formed in 25 patients, being positive in 9 (36%) patients. Amyloid deposition was present in septa and vascular walls, but it was often scant and difficult to detect. Skin Biopsy All of the skin specimens (16 patients) were negative. No traces of amyloid could be observed in any of the cutaneous structures. Rectal Biopsy Rectal biopsy was performed in 12 patients. Positive results were obtained in 3 out of the 7 patients in whom the sample included submucosa (43%). In the other 5 negative samples, no submucosal tissue was obtained; therefore, amyloid infiltration could not be excluded. Two-Dimensional Echocardiogram Bidimensional echocardiography was performed in 23 patients. The échocardiographie examination of 12 (52%) out of the 23 patients studied was suggestive of cardiac amyloidosis, given the presence of nodular struc tures of the interventricular septum (‘granular spark ling') and increased atrial septum thickness. Surgical Specimens Stomach (n = 1). A massive and diffuse deposit of amyloid substance was found mainly in the external layer of muscularis propria (fig. 1). Also vascular subendothelial deposits were seen in the submucosa and in the muscular gastric wall.
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Sigmoid Cohn (n = 2). In both cases histological examination showed nodular subendothelial amyloid deposits in arterioles and small arteries of the intestinal submucosa (fig. 2), leading to a total or subtotal obstruc tion of the arteriolar lumen in some vessels. Focal depo sition in muscularis propria was observed in 1 case. Autopsies o f Amyloidotic Patients Patient No. 2. A 60-year-old man on hemodialysis during the last 11 years for chronic renal failure second ary to nephrosclerosis. Clinically bilateral amyloid car pal tunnel syndrome was observed. He died from a lung carcinoma. Necropsy showed nodular amyloid deposits in the subendothelium of arterioles and small arteries of intestinal submucosa, heart, liver, lungs, testicles, and adrenal glands. Moreover, diffuse deposits were found in heart, prostate, and perithyroid tissue. No amyloid de posits were found in kidney, pancreas, spleen, and cen tral nervous system. Patient No. 10. A 72-year-old women on maintenance hemodialysis since 1977 because of a chronic interstitial nephritis. In 1983 she underwent release of a right carpal tunnel syndrome, demonstrating amyloid substance in the surgical specimen. In 1984. she complained of pain ful persistent effusion and swelling in knees and should ers, and amyloid origin was confirmed by a synovial biopsy. In 1986 the patient was admitted because of a pathological fracture of the left femur neck. Massive amyloid infiltration was observed in the fractured bone. A biopsy of the subcutaneous abdominal fat was per formed and proved to be amyloid positive. A twodimensional echocardiogram showed nodular images in
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Fig. 2. Subcndothclial amyloid deposit in the arteriolar wall of rectal submucosa, reacting strongly with fL-micoglobulin antiserum. Avidin-biotin-peroxidase complex. X 300.
the interventricular septum, suggestive of cardiac amy loidosis (fig. 3a). Rectal and skin biopsies discarded the presence of amyloid substance. The patient died in April 1988 secondary to an aluminum encephalopathy. Nec ropsy discarded visceral involvement, except for the presence of nodular amyloid deposits (3-5 mm in diam eter) in the myocardium (fig. 3b). Immunohistochemical examination confirmed the (^-microglobulin origin of the myocardial amyloid deposits. Patient No. 26. A 62-year-old woman affected by chronic renal failure secondary to polycystic kidney dis ease and on maintenance hemodialysis during the last 13 years. Two years prior to death, she underwent surgery for a left carpal tunnel syndrome, and amyloid substance was found in the transverse carpal ligament. One year later, she complained of right-knee pain, with swelling and effusion, and amyloid substance was found in the synovial fluid sediment. Subcutaneous abdominal fat aspiration was positive for amyloid deposits. Two-di mensional echocardiography excluded cardiac involve ment. The patient died from a Legionella pneumonia. Necropsy showed nodular amyloid deposits in the sub endothelium of arterioles and small arteries of the intes tinal submucosa. Also amyloid deposits were confirmed in the osteoarticular structures. The rest of the visceral specimens were negative for Congo red stain.
massive amyloid deposits in osteoarticular structures, with severe involvement of synovial membrane and cap sules of shoulders and knees. No amyloid substance was demonstrated in visceral tissues. Necropsy was negative (visceral and bone tissues) for amyloid deposition in the remaining 4 asymptomatic patients. The rate of visceral involvement was statisti cally highly sinificant in the symptomatic patient group (p < 0.02) and clearly related to the time on hemodialy sis (p < 0.0001; table 2).
Autopsies o f Asymptomatic Patients A 72-year-old man with nephrosclerosis and on main tenance hemodialysis during the last 4 years. He had no related clinical manifestations of dialysis amyloidosis. He died from myocardial infarction. Necropsy showed
Pathology Studies Amyloid substance exhibited the characteristic stain with Congo red. Following treatment with potassium permanganate a partial loss of congophilia was observed, but green birefringence was clearly maintained.
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Fig. 3. a Left: two-dimensional echocardiogram showing nodular structures at the interventricular septum ('granular sparkling'; ar rows), suggestive of cardiac amyloidosis. VD = Right ventricule; VI = left ventricule; AD = right auricule; AI - left auricule. b Right: nodular deposition of amyloid substance in the myocardium. Congo red. X 20.
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(L-Microglobulin was positive in all tested tissues. Areas with positive reactions corresponded well with Congo red positive deposits in parallel sections. All the specimens used as controls were negative. Amyloid P component was positive in nearly all tested samples, including cardiac and intestinal deposits.
Discussion Dialysis amyloidosis has recently been recognized as a new complication in the osteoarticular area in long term hemodialyzed patients. The first description of this new type of amyloidosis was in 1980, when Assenat et al. [35] demonstrated the amyloid origin of the carpal tun nel syndrome in hemodialyzed patients. Afterward, in 1984, Charra et al. [36] reported the high association of amyloid carpal tunnel syndrome with shoulder pain in patients on hemodialysis. Dialysis amyloidosis involves predominantly osteoarticular structures, and it has been found most often to be associated with destructive ar thropathy, destructive spondyloarthropathy, pathologi cal fractures, and carpal tunnel syndrome [1-14], In par ticular, it has been detected in synovium, capsula, sub chondral bone, and in intervertebral disks. Gejyo et al. [19] and Shirahama et al. [20] in 1985 identified the p2 microglobulin as the major constituent protein of this new type of amyloidosis. The question whether such amyloidosis is localized to wrists and contiguous structures, confined to the muscu loskeletal system, or widely disseminated has remained controversial. The extent of organ involvement due to amyloid may have prognostic significance for these pa tients. At present, marked extra-articular deposits have been only reported sporadically. However, some faint p2 microglobulin amyloid deposition has been demon strated in the wall of arterioles of several visceras, including heart, lungs, liver, and intestine [20, 21, 2326, 37-39], In all these reported patients, the visceral involvement was asymptomatic except in 3 cases re ported by Fuchs et al. [22] who developed gastrointesti nal bleeding associated with vascular amyloid deposits in one and odynophagia associated with lingual amyloid infiltration in the other and in the case reported by Shinoda et al. [39] with intestinal pseudo-obstruction. Systemic studies have failed to show amyloid deposits in abdominal fat aspiration and in skin biopsy [12. 26, 32]. Recently, Varga et al. [40] have reported the lack of amyloid in abdominal fat aspirates from 30 patients undergoing long-term hemodialysis, although only 11
patients complained of carpal tunnel syndrome and nei ther of them with histological confirmation of P2-microglobulin amyloid origin. Altemeyer et al. [29] in 1983 suspected a high incidence of skin amyloid deposits in hemodialyzed patients, but revised their view later, stat ing that the Congo red positive material detected in the corium of the skin of dialized patients was most proba bly altered collagen [41], Rectal biopsy specimens in patients affected by dialysis amyloidosis have shown small perivascular amyloid deposits on the submucosa layer in 6 of 9 reported patients, but none of these patients had clinically significant amyloid involvement [2, 20, 25]. Liver biopsy performed once in a dialyzed patient with a P2-microglobulin amyloidosis was amy loid negative [6], Hawkins et al. [42], using l23I-labelled purified human serum amyloid P component, failed to find visceral deposits in 2 hemodialysis patients affected by P2 -microglobulin amyloidosis. We have previously reported visceral involvement in 3 hemodialyzed patients with amyloid deposits in the wall of the vessels of several viscera and demonstrated the P2 -microglobulin origin [21]. We then started a pro spective study to confirm the systemic character of dial ysis amyloidosis. The study, included 26 of our 35 patients with dialysis amyloidosis, in whom at least two systemic examinations were performed. Also, five nec ropsies of dialyzed patients without symptomatic dialy sis amyloidosis were included. In 15 of 26 patients (58%) amyloid deposits were histologically demonstrated in visceral structures and in 21 of these patients (81 %), if the positive two-dimensional echocardiograms were considered. Whereas the five necropsies of asymptom atic patients failed to demonstrate visceral involvement, and although none of the patients had had articular symptoms, one of them showed osteoarticular infiltra tion. None of the patients with visceral deposits had sys temic clinical manifestations. This higher incidence of visceral involvement in our patients could be due to sev eral factors: the long time on hemodialysis (mean 12 years), the advanced age (mean 60 years), and the selected group of studied patients, all of them affected by symptomatic dialysis amyloidosis. Abdominal fat aspiration represents for most groups the best diagnostic procedure in the evaluation of sys temic amyloid disease [28]. Up to now. all abdominal fat samples of dialyzed patients were negative for (^-microglobulin amyloid, including our first description of amy loid arthropathy in long-term hemodialysis patients in which six abdominal fat specimens were amyloid nega tive [1, 23, 38]. In the present study, amyloid was present
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Systemic Involvement of Dialysis-Amyloidosis
involvement (carpal tunnel syndrome and amyloid ar thropathy) and is followed by the visceral one with pro gressive nodular subendothelial amyloid infiltration. In future, with the increased age of the dialyzed population, visceral involvement would be more usual and presum ably with systemic symptoms. Acknowledgments This work was supported in part by grant CIRIT-AR87 and has been presented as a free communication at the Xth International Congress of Nephrology, London. England, July 1987. and at the XXIVth Congress of the EDTA-Europcan Renal Association, Berlin (West). October 1987.
References 1 Muñoz-Gómez J, Bergada E, Gómez R. Llopart E, Subías E, Rotes J, Solé M: Amyloid arthropathy in patients undergoing periodical haemodialysis for chronic renal failure: A new com plication. Ann Rheum Dis 1985;44:729-733. 2 Morita T. Suzuki M. Kamimura A, Hirasawa Y: Amyloidosis of a possible new type in patients receiving long-term hemodialysis. Arch Pathol Lab Med 1985;109:1029-1032. 3 Bardin T, Kuntz D. Zingraff J. Voisin MC. Zelmar A. Lansaman J: Synovial amyloidosis in patients undergoing long-term hemo dialysis. Arthritis Rheum 1985:28:1052—1058. 4 Brown EA. Arnold JR, Gower PE: Dialysis arthropathy: Compli cation of long-term treatment with hemodialysis. Br Med J 1986; 292:163-166. 5 Hardouin P, Flipo RM. Foissac-Gegoux P, Thevcnon A. Pouyol F. Duquesnoy B. Delcambre B: Current aspects of osteoarticular pathology in patients undergoing hemodialysis: Study of 80 patients. I. Clinical and radiological analysis. J Rheumatol 1987: 14:780-783. 6 Fenvcs A. Emctt M. White M. Greenway G. Michaels D: Carpal tunnel syndrome with cystic bone lesions secondary' to amyloid osis in chronic hemodialysis patients. Am J Kidney Dis 1986;7: 130-134. 7 Bergada E. Montoliu J. Subias R. Sole M. Lopez-Pedret J, Revert LL: Síndrome del túnel carpiano con deposito local y articular de sustancia amiloide en el hemodializado. Med Clin (Bare) 1986:86:319-322. 8 Huaux JP. Noël H. Bastien P. Malghem J. Maldaguc B. Dcvogelaer JP. Nagant de Deuxchaisnes C: Amvlose articulaire, fracture du col fémoral et hémodialyse périodique chronique. Rev Rhum Mal üstéoartic 1985:52:179-182. 9 Muñoz-Gómez J. Gómez R. Llopart E. Solé M: The clinical pic ture of amyloid arthropathy in patients with chronic renal fail ure maintained on hemodialysis using cellulose membranes. Ann Rheum Dis 1987:46:573-579. 10 Hadjipavlou A. Lander P, Beg:n L. Bercovitch D. Davidman M. Jakab E: Skeletal amyloidosis due to beta microglobulinemia in a patient on hemodialysis. J Bone Joint Surg 1988:70:119-121. 11 Schwarz A. Keller F. Scyfcrt S. Poll W, Molzahn M. Distler A: Carpal tunnel syndrome: A major complication in long-term hemodialysis patients. Clin Nephrol 1984;22:133-137.
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in septa and vascular wall of 9 out of the 26 abdominal fat specimens (36%); 3 of them were previously negative. Two-dimensional echocardiography represents a useful tool to define cardiac involvement in amyloid diseases, if increased atrial septal thickness with increased myo cardial echogenicity (‘granular sparkling’) is present [31], In our study, two-dimensional echocardiography was the most sensitive method to define visceral involvement and was suggestive of cardiac amyloidosis in 12 out of 23 patients studied (52%). Unfortunately, endocardium biopsy is too aggressive to practice routinely, but we have confirmed the presence of a positive echocardio gram and cardiac involvement in the necropsy of 1 patient (No. 26) who presented nodular amyloid depos its in the myocardium. There are no previous reports of two-dimensional echocardiography in p2-microglobulin amyloidotic patients, and we consider that more experi ence would be necessary to confirm our findings. Skin biopsy was negative for amyloid in all 16 speci mens, so we think that it is not a useful tool in this type of amyloidosis. Rectal biopsy was performed in 12 pa tients and demonstrated the presence of nodular suben dothelium amyloid deposits in arterioles and small arter ies of the rectal submucosa in three specimens. Amyloid infiltration was only shown in submucosa vessels; there fore, to investigate dialysis amyloidosis, rectal biopsy must be deep enough to contain rectal submucosa. In the surgical specimens (1 stomach and 2 colon) amyloid substance was demonstrated in all examina tions. Amyloid deposits fitted in two different patterns: in a diffuse form infiltrating the submucosa layer and in nodular subendothelial deposits in the vascular wall of arterioles and small arteries. In the three autopsies of dialysis amyloidosis patients visceral involvement was clearly demonstrated in all, with nodular subendothelial amyloid deposits in arteri oles of several organs and nodular deposits in endocar dium and myocardium. Amyloid substance reacted strongly with anti-p2-microglobulin and not with the other antisera, confirming the exclusively p2-microglobulin origin. Amyloid infiltration was demonstrated only in 1 of the 5 autopsies of the asymptomatic patients and only in osteoarticular structures and not in viscera. In summary, we think that dialysis amyloidosis has a systemic character with visceral involvement. Two-di mensional ehocardiography represents the most useful tool to search for the visceral involvement of p2-microglobulin amyloidosis, followed by abdominal fat aspira tion and rectal biopsy. The natural course of this new type of amyloidosis goes first with the osteoarticular
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12 DiRaimondo CR. Casey TT, DiRaimondo CV, Stone WJ: Pathologic fractures associated with idiopathic amyloidosis of bone in chronic hemodialysis patients. Nephron 1986;43:22— 27. 13 Csey TT. Stone WJ, DiRaimondo CR. Page DL, Gorevic PD: Dialysis-related amyloid is amyloid of beta-2-microglobulin (AM b2m) origin. Arthritis Rheum 1986;29:1170. 14 Zingraff J, Bardin T, Kuntz D, Voisin MC, Juquel JP, Driieke T: Degenerative osteo-articular lesions and amyloid infiltration in long-term hemodialysis patients. Proc Eur Dial Transplant As soc Eur Renal Assoc 1985;22:131-135. 15 Allain TJ, Stevens PE, Bridges LR. Phillips ME: Dialysis my elopathy: Quadriparesis due to extradural amyloid of (L-microglobulin origin. Br Med J 1988;296:752-753. 16 Kuntz D, Naveau B, Bardin T, Driieke T, Treves R, Dryll A: Destructive spondyloarthropathy in hemodialyzed patients: A new syndrome. Arthritis Reum 1984;27:369-375. 17 Muñoz Gomez J, Estrada P: Early radiologic manifestations of destructive spondylarthropathy in hemodialyzed patients. Ar thritis Reum 1986;29:1171-1172. 18 Sebert JL, Fardellone P, Marie A, Deramond H, Lambrey G, Legars D, Galibert P, Smajda A, Fournier A: Destructive spondylarthopathy in hemodialyzed patients. Possible role of amy loidosis. Arthritis Rheum 1986;29:301-303. 19 Gejyo F, Yamada T, Odani S, Nakagawa Y, Arakawa M, Kunitomo T, Kataoka H, Susuki M, Hirasawa Y, Shirahama T, Cohen AS, Schmid K: A new of amyloid protein associated with chronic hemodialysis was identified as (ii-microglobulin. Biochem Biophys Res Commun 1985;129:701-706. 20 Shirahama T, Skinner M, Cohen AS, Gejyo F, Arakawa M, Suzuki M, Hirasawa Y: Histochemical and immunohistochemi cal characterization of amyloid associated with chronic hemodi alysis as (îi-microglobulin. Lab Invest 1985;53:705-709. 21 Campistol JM, Cases A, Torras A, Muñoz-Gómez J, Solé M. Montoliu J, Lopez-Pedret J, Revert LL: Visceral involvement of dialysis amyloidosis. Am J Nephrol 1987;7:390-393. 22 Fuchs A, Jagirdar J, Schwartz IS: Beta-2-microglobulin amyloid osis (AB2M) in patients undergoing long-term hemodialysis. Am J Clin Pathol 1987;88:302-307. 23 Noël LH, Zingraff J, Bardin T, Atienza C, Kuntz D Driieke T: Tissue distribution of dialysis amyloidosis. Clin Nephrol 1987; 27:175-178. 24 Ogawa H, Saito A, Hirabayashi N, Hara K: Amyloid deposition in systemic organs in long-term hemodialysis patients. Clin Nephrol 1987;28:199-204. 25 Hardouin P, Flipo RM, Lecomte-Houcke M, Foissac-Gcgoux P, Delcambre B: Amylose des hémodialyses. Recherche d’une dif fusion générale par biopsie rectale. Presse Méd 1987; 16:445446. 26 Theaker JM. Raine AEG, Rainey AJ, Heryet A, Clark A, Oliver DO: Systemic amyloidosis of P;-microglobulin type: A complica tion of long-term hemodialysis. J Clin Pathol 1987;40:1247— 1251. 27 Muñoz-Gómez J, Gomez Perez R, Sole M, Llopart E: Synovial fluid examination for the diagnosis of synovial amyloidosis in patients with chronic renal failure undergoing hemodialysis. Ann Rheum Dis 1987;46:324-326. 28 Duston MA, Skinner M. Shirahama T, Cohen AS: Diagnosis of amyloidosis by abdominal fat aspiration. Analysis of four years’ experience. Am J Med 1987;82:412-414.
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29 Altemeyer P. Kachel HG, Runne U: Microangiopathy, altera tions of connective tissue, and deposition of an amyloid-like substance in patients with chronic renal failure. Hautarzt 1983; 34:277-285. 30 Siqueira-Filho AG, Cunha CLP, Tajik AJ, Seward JB, Schatten berg TT, Giuliani ER: M-mode and two-dimensional échocar diographie features in cardiac amyloidosis. Circulation 1981 ;63: 188-196. 31 Falk R. Plchn J, Deering T. Schick EC, Boinay P, Rubinow A, Skinner M, Cohen AS: Sensitivity and specificity of échocardio graphie features of cardiac ayloidosis. Am J Cardiol 1987;59: 418-422. 32 Janssen S, Van Rijswijk MH, Meijer S, Ruinen L, Van der Hem GK: Clinical evaluation of amyloid arthropathy and AL amyloid disease; in Marrink J, Van Rijswijk MH (eds): Amyloidosis. Dordrecht, Nijhoff, 1986, pp61-72. 33 Wright JR, Calkins E, Humphrey RL: Potassium permanganate reaction in amyloidosis. A histologic method to assist in differ entiating forms of this disease. Lab Invest 1977;36:274-281. 34 Hsu SM, Raine L, Fänger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelcd antibody (UAP) procedures. J Histochem Cytochem 1981;29:577-580. 35 Assenât U, Calemard E, Charra B, Laurent G, Terrât JC, Vanel T: Hémodialyse syndrome du canal carpien et substance amy loïde. Nouv Presse Méd 1980;9:1715. 36 Charra B, Calemard E, Uzan M, Terrât JC, Vanel T, Laurent G: Carpal tunnel syndrome, shoulder pain and amyloid deposits in long-term hemodialysis patients. Proc Eur Dial Transplant As soc Eur Renal Assoc 194;21:291-295. 37 Takahashi S, Morita T, Koda Y, Murayama H, Hirasawa Y: Gastrointestinal involvement of dialysis-related amyloidosis. Clin Nephrol 1988;30:168-171. 38 Floege J. Brandis A, Nonnast-Daniel B. Westhoff-Bleck M, Tiedow G, Linke RP, Koch KM: Subcutaneous amyloid tumor of beta-2-microglobulin origin in a long-term hemodialysis patient. Nephron 1989;53:73-75. 39 Shinoda T, Komatsu M, Aizawa T, Shirota T, Yamada T, Ehara T, Mizukami E: Intestinal pseudo-obstruction due to dialysis amyloidosis. Clin Nephrol 1989;32:284-289. 40 Varga J, Idelson BA, Felson D, Skinner M, Cohen AS: Lack of amyloid in abdominal fat aspirates from patients undergoing long-term hemodialysis. Arch Intern Med 1987; 147:14551457. 41 Kachel HG, Altemeyer P, Kühn KW, Koch KM. Baldamus CA: Deposition of non-amyloid material in connective tissue in urae mia. Blood Purif 1984;2:142-144. 42 Hawkins PN, Myers MJ, Lavender JP, Pepys MB: Diagnostic radionuclide imaging of amyloid: Biological targeting by circu lating human serum amyloid P component. Lancet 1988;i: 1413— 1418. Received: December 8, 1989 Accepted: April 11,1990 Dr. J.M. Campistol Nephrology Service Hospital Clinic i Provincial 170, Villarroel E-08036 Barcelona (Spain)
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