Coexistence of Protein AA and Immunoglobulin Light-Chain Fragments in Amyloid Fibrils

Coexistence of amyloid fibril protein AA and homogeneous immunoglobulin light-chain fragments was found in the isolated amyloid fibrils of two patients with amyloidosis secondary to rheumatoid arthritis. The light-chain amyloid fibril protein showed antigenic identity with a light-chain amyloid from a patient with primary amyloidosis, which was identified as the VlambdaIV subgroup by amino acid sequence analysis. In the amyloid fibrils isolated from another patient with primary amyloidosis there was a mixture of VlambdaIV and VlambdaV homogeneous immunoglobulin light chains. Thus, a mixture of protein AA had lambda light chains or two different types of homogeneous light chains may be found in the amyloid fibrils of some patients.     

Lmmunohistochemical classification of 140 autopsy cases with systemic amyloidosis

One hundred and forty autopsy cases of systemic amyloid-osis were examined using the potassium permanganate method for distinction of amyloid A protein from other amyloid proteins and an immunohistochemical technique. Of those cases, amyloid proteins were identified in 121 cases. There were 68 cases of amyloid A-related (AA) amyloidosis and these were the most common type among the cases (56.2%). There were 39 cases of immunoglobulin light chain-related (AL) amyloidosis (32.2%), six cases of β₂-microglobulin-related (Aβ2M) amyloidosis (5%), and five cases of transthyretin-related (ATTR) amyloidosis (4.1%). Minute areas of amyloid deposits in four cases with AA were resistant to potassium permanganate pretreatment. In Aβ2M amyloidosis amyloid deposits were either resistant or sensitive to potassium permanganate pretreatment, from case to case. The coexistence of two different amyloid proteins was seen in three cases: one case had ATTR and Ax types, and two cases had Aβ2M and AA types. Some discrepancies were seen between the immunohistochemical typing and clinical classification of amyloidosis referred to in the Annual of the Pathological Autopsy Cases in Japan, for example, one case of AA type in myeloma-associated amyloidosis and one case of AL type in secondary amyloidosis. From the present results, the importance of the immunohistochemical method in classifying amyloidosis is stressed.     

The potassium permanganate method. A reliable method for differentiating amyloid AA from other forms of amyloid in routine laboratory practice.

Alterations in affinity of amyloid for Congo red after incubation of tissue sections with potassium permanganate, as described by Wright el al, were studied. The affinity of amyloid for Congo red after incubation with potassium permanganate did not change in patients with myeloma-associated amyloidosis, familial amyloidotic polyneuropathy, medullary carcinoma of the thyroid, pancreatic island amyloid, and cerebral amyloidosis. Affinity for Congo red was lost after incubation with potassium permanganate in tissue sections from patients with secondary amyloidosis and amyloidosis complicating familial Mediterranean fever (consisting of amyloid AA). Patients with primary amyloidosis could be divided into two groups, one with potassium-permanganate--sensitive and one with potassium-permanganate--resistant amyloid deposits. These two groups correlated with the clinical classification in typical organ distribution (presenting with nephropathy) and atypical organ distribution (presenting with cardiomyopathy, nephropathy, and glossopathy) and the expected presence of amyloid AA or amyloid AL. Potassium permanganate sensitivity seems to be restricted to amyloid AA. The potassium permanganate method can be important in dividing the major forms of generalized amyloidosis in AA amyloid and non-AA amyloid. This can be used for differentiating early stages of the disease and cases otherwise difficult to classify. It is important to define patient groups properly, especially in evaluating the effect of therapeutic measures. (Am J Pathol 97:43--58, 1979).     

Characterization of different amyloids with immunological techniques

We studied 71 cases of amyloidosis from the autopsy material of our institute. 17 cases were secondary amyloidosis of which 7 had initially been diagnosed as primary amyloidosis; all cases reacted positively with an anti-substance A antibody; 13 showed a suppression and 4 a strong reduction of the Congo red staining following tissue incubation in KMnO4. 25 cases were identified as primary amyloidosis of which 7 had initially not been recognized as such. In 16 cases amyloid deposits reacted positively with antibodies specific for the light chains of the immunoglobulins (3 X kappa, 13 X lambda). A monoclonal plasmacytic cell proliferation in the bone marrow was seen in 14 cases. In all cases deposits were KMnO4 resistant, but 1 case showed a slight reduction of staining intensity. 27 cases were cardio-vascular (senile) amyloidosis; in 20 cases at least 3 organs showed deposits; 12 cases had deposits in 5 and more organs. 2 cases were heredofamilial amyloidosis. In those 29 cases deposits reacted positively with an anti-prealbumin antibody, but were negative for AA and the light chains of the immunoglobulins; the Congo red staining remained strong in all cases when previously incubated in KMnO4. KMnO4-Congo red staining and antisera specific for AA, L-chains and prealbumin proved of value for classification of amyloidosis and for its organ distribution.     

The pattern of amyloid deposition in the lung

A review of routine histopathological samples and autopsies examined at the Department of Pathology, University of Malaya revealed 15 cases of amyloidosis of the lung. Two were localized depositions limited to the lung while in the remainder, lung involvement was part of the picture of systemic amyloidosis. Both cases of localized amyloidosis presented with symptomatic lung/bronchial masses and a clinical diagnosis of tumour. Histology revealed "amyloidomas" associated with heavy plasma cell and lymphocytic infiltration and the presence of multinucleated giant cells. In both cases, the amyloid deposits were immunopositive for lambda light chains and negative for kappa chains and AA protein. One was a known systemic lupus erythematosus patient with polyclonal hypergammaglobulinaemia. The other patient was found to have plasma cell dyscrasia with monoclonal IgG lambda gammopathy. Both patients did not develop systemic amyloidosis. In contrast, lung involvement in systemic AA amyloidosis was not obvious clinically or macroscopically but was histologically evident in 75% of cases subjected to autopsy. Amyloid was detected mainly in the walls of arterioles and small vessels, and along the alveolar septa. It was less frequently detected in the pleura, along the basement membrane of the bronchial epithelium and around bronchial glands. In one case of systemic AL amyloidosis associated with multiple myeloma, an "amyloidoma" occurred in the subpleural region reminiscent of localized amyloidosis. These cases pose questions on (1) whether localized "tumour-like" amyloidosis is a forme fruste of systemic AL amyloidosis and (2) the differing pattern of tissue deposition of different chemical types of amyloid fibrils, with the suggestion that light chain amyloid has a greater tendency to nodular deposition than AA amyloid.     

Diagnosis and immunohistochemical classification of systemic amyloidoses

 Fourty-three cases of systemic amyloidosis were identified in an unselected autopsy series from our institute (6305 autopsies between 1979 and 1993) and classified immunohistochemically by means of a panel of antisera directed against five major amyloid fibril proteins. Amyloid A (AA) amyloidosis was the most common type, being found in 21 cases (48.8%). Transthyretin-derived (ATTR) amyloidosis was present in 11 cases (25.6%), and immunoglobulin light chain-derived (AL) amyloidosis in 10 cases (23.3%). A single case (2.3%) contained deposits of more than one type of systemic amyloid. AA amyoloidosis was associated with chronic inflammatory or infectious diseases (81%), malignant tumours (19%) or both (9.5%). Immunoglobulin light chain-derived amyloidoses were associated with myeloma (50%) or primary (idiopathic; 50%). In AA and AL amyloidosis the kidney was the organ most frequently involved. ATTR amyloid affecting mostly the heart and lungs presented as senile systemic amyloidosis. Systemic amyloidosis was the cause of death in 5 cases (12%) and caused symptoms in 17 cases (39%). Our results suggest that most cases can be classified by using a panel of sensitive and specific antibodies against five major amyloid fibril proteins. This technique may make amyloid type-specific therapy possible for AL amyloid patients who do not have evidence of an underlying plasma cell dyscrasia. Received: 9 December 1997 / Accepted: 2 February 1998     

Immunofluorescence study of "non-idiopathic" renal amyloidosis

The authors report the results of immunofluorescence (IF) studies of 17 cases of "non-idiopathic" renal biopsy-proven amyloidosis and 18 cases of various nephropathies and normal kidneys (as controls), investigated by IF by simultaneous use of antisera against routine IgG, IgM, IgA, C3, C4, Clq, beta-lipoprotein, albumin, and fibrinogen. Antisera against kappa and lambda light chains and amyloid A and amyloid P components were also used. Six of the 17 cases of amyloidosis were associated with immunocyte dyscrasia, and 11 were cases of reactive systemic amyloidosis associated with chronic infections or inflammatory and neoplastic disorders. In amyloidosis, IF deposits appeared for all antisera as homogeneous staining of mesangial nodules, and, more rarely, there was staining along the glomerular basement membranes. Overall immunoglobulins and C3 were present in 11 cases (64 per cent). Kappa and lambda light chains were demonstrated in 14 (82 per cent) and 12 (70 per cent) cases, respectively. In immunocyte dyscrasia associated with amyloidosis, immunoglobulin and light-chain deposits corresponding to a paraprotein abnormality were demonstrated in glomeruli and in tubular casts. Amyloid P component was always present in glomeruli with a bright and characteristic fluorescence, and it was frequently observed in arterioles. Amyloid A component was observed in six cases of reactive systemic amyloidosis but also in one case of immunocyte dyscrasia with amyloidosis. In view of the diversity of amyloid fibril types and their chemical nature, IF studies confirm the presence of different constituents but do not warrant any conclusion concerning the pathogenesis of this disease.     

Tumoral amyloidosis of bone of beta 2-microglobulin origin in association with long-term hemodialysis: a new type of amyloid disease

Amyloid lesions of bone are rare and limited almost exclusively to patients with amyloidosis secondary to plasma cell dyscrasias. The present report describes the cases of two patients receiving long-term hemodialysis (nine and 12 years) who had multiple lytic lesions of bone proved by biopsy to contain an unusual type of amyloid. Results of serum protein electrophoreses and immunoelectrophoreses, as well as bone marrow examinations, were normal. In both cases the amyloid displayed characteristic Congo red affinity and birefringence on polarized light microscopy that was inhibited by potassium permanganate treatment of sections prior to staining. Although this staining reaction was described previously exclusively in AA amyloid (i.e., the material associated with classic secondary amyloidosis), immunoperoxidase staining for AA protein in these cases was negative. Transmission electron microscopy revealed the amyloid fibrils to have unusual curvilinear configurations. Immunoperoxidase staining for beta 2-microglobulin (beta 2m) was positive in the amyloid lesions of both patients at the light microscopic level. Ultrastructural immunohistochemical studies for beta 2m, performed in one case, were positive. Both patients had markedly elevated serum beta 2m levels. By Ouchterlony immunodiffusion, purified beta 2m demonstrated partial identity with purified amyloid protein fractions and a serum constituent. Bone lesions composed of amyloid related to beta 2M probably represent a new subgroup of amyloid disease that may be linked to renal failure and long-term hemodialysis.     

Protein AA in primary and myeloma associated amyloidosis.

Protein AA, the main fibril constituent in secondary systemic amyloidosis, was demonstrated by the peroxidase-anti-peroxidase method in kidney sections from five out of 14 cases of primary and myeloma associated amyloidosis, all having an immunoglobulin light chain derived protein as a major subunit. In three of these cases, protein AA was also demonstrated in double immunodiffusion of dissolved amyloid preparations. The protein had the characteristics of protein AA in elution position, immunodiffusion and isoelectric focussing pattern. The significance of protein AA in primary and myeloma associated amyloidosis is unknown.     

Immunoglobulin synthesis in primary and myeloma amyloidosis.

Bone marrow cells from 14 patients with primary amyloidosis and two patients with myeloma amyloidosis were studied by immunofluorescence and biosynthesis experiments after incorporation of radioactive amino acids. Cells from four patients affected with non-myeloma secondary amyloidosis were also studied as controls. In primary amyloidosis, monoclonal plasma cell populations were demonstrated by immunofluorescence in virtually every case, even in patients without serum and urine monoclonal immunoglobulin and with a normal percentage of bone marrow plasma cells. Biosynthesis experiments showed the secretion of large amounts of free light chains, most often of the lambda type, in every primary or myeloma amyloidosis case and the presence of light chain fragments in almost all cases. Special features in certain patients were the synthesis of short gamma chains (two cases), assembly block at the HL half molecule level of a monoclonal IgA (one case) and secretion of decameric abnormally large kappa chains (one case). This is in contrast with non-myelomatous secondary amyloidosis where the distribution of bone marrow plasma cells was normal by immunofluorescence and where normal sized immunoglobulins were synthesized, without free light chain secretion and fragments. These data confirm that primary amyloidosis belongs to plasma cell dyscrasias and emphasize the role of free light chains and light chain fragments in the pathogenesis of amyloid deposition.     

Morphologic differences in the pattern of liver infiltration between systemic AL and AA amyloidosis

Biopsy and necropsy tissue from 31 unselected patients with systemic amyloidosis, in which there was histologic evidence of liver involvement, were reviewed with reference to the location and pattern of amyloid deposition in the liver. Amyloidosis was classified into AA and AL types on the basis of immunohistochemistry and permanganate reaction of the amyloid deposits. Nineteen were categorized as AA (secondary) and 12 as AL (primary) amyloidosis. Deposition of AA amyloid was limited to the walls of vessels in the portal tract, constituting a "vascular" pattern. In AL amyloidosis, the deposits exhibited a "sinusoidal" pattern in that they were seen along hepatic sinusoids as well as in vessel walls. This difference was statistically significant (P less than .001). The histologic pattern of liver infiltration offers a valuable clue in the classification of systemic amyloidosis and provides information that may be useful in the selection of patients for therapy.     

Immunohistochemical characterization of renal amyloidosis

Forty-five renal biopsies with amyloidosis were studied by light microscopy with Congo red staining and action of potassium permanganate and by immunofluorescence with antihuman tissue A component antiserum antilight and heavy chains of immunoglobulins antisera. The patients were classified on the basis of concordance between immunohistochemical characterization by immunofluorescence and the results of Congo red staining after potassium permanganate treatment. Thus, 37 of 45 cases (82%) were classified by immunohistochemical characterization (15 with AL amyloidosis and 22 with AA amyloidosis) when the amyloid type could be hypothetized in only 31 of these cases (66%) on the basis of clinical criteria. This study suggests that the association of these two technics is more reliable than clinical data alone in distinguishing between AA and AL amyloidosis.     

Immunohistochemical characterization of amyloid proteins in sural nerves and clinical associations in amyloid neuropathy.

To test whether immunohistochemical characterization of proteins in amyloid deposits in biopsied sural nerves gives reliable and useful diagnostic information using commercially available reagents, biopsy specimens of sural nerves from 38 patients with amyloid neuropathy were studied. Transthyretin (TTR) was detected in the amyloid deposits of 11 nerves, lambda light chains (LC) in 8 nerves, kappa LC in 7 nerves, and both lambda and kappa LC in 3 nerves. In 9 nerves, the amyloid deposits were too small to allow adequate immunohistochemical characterization of amyloid proteins in serial sections. Evidence that immunohistochemical characterization was correct came from: 1) evaluation of kin, 2) search for monoclonal proteins in the plasma, and 3) sequencing of the gene abnormalities in TTR+ cases. In 9 of 11 TTR+ cases, in which DNA could be obtained, sequencing of the gene showed that each of the 9 cases was heterozygous for a gene mutation; 7 had previously described mutations and 2 undescribed mutations. Therefore, in the nine sporadic cases without plasma monoclonal light chains, the immunohistochemical characterization correctly identified the protein in amyloid as transthyretin. Likewise, there was a high concordance between immunoglobulin light chains in plasma and light chains in amyloid in primary amyloidosis. Evaluation of the type, distribution, and severity of the neurologic symptoms and deficits showed: 1) the sensorimotor and autonomic neuropathy of amyloidosis characteristically affects proximal as well as distal limbs, and 2) the type of amyloidosis probably cannot be determined from the characteristics or severity of the neuropathy alone or from the location or size of amyloid deposits in nerve.     

Senile amyloid deposition

Amyloid deposition in the aged was studied in a wide selection of tissues from 194 necropsies of subjects aged over 65 years, there being no clinical manifestation of amyloidosis. We employed conventional Congo red staining with the aid of a polarising filter for the identification of amyloid. We analysed the deposits histochemically for tryptophan content and reaction to oxidation using the potassium permanganate method in 26 subjects aged over 85 years. Both the frequency of amyloid deposition and the number of organs involved tended to increase with advancing age, and both increased after the ninth decade with little sex difference. Amyloid deposition preferentially occurred in the aorta, heart, lung, prostate and pancreas among the organs examined. Visceral involvement tended to be confined to the vasculature except in the prostate and pancreas. In cases associated with predisposing conditions considered to evoke secondary amyloidosis such as tuberculosis, multiple myeloma, and rheumatoid arthritis more amyloid was found but the distribution of the deposits was similar by organ and intravisceral site to that of cases unassociated with predisposing disorders. In both groups of cases amyloid was found to contain tryptophan, indicative of immunoamyloid and, with a few exceptions, to be resistant to oxidation by potassium permanganate indicating an immunoglobulin AL component. It can be concluded that senile amyloid should be classified as a form of so-called primary amyloidosis because of its widespread distribution, its occurrence independent of predisposing disorders and its chemical composition. Subclinical chronic inflammation of the respiratory, gastrointestinal and urogenital tracts may provide the necessary prolonged immunological stimulus to lead to amyloid deposition in ageing tissues.     

Senile aortic amyloid. A third distinctive type of age-related cardiovascular amyloid.

Aortic tissues from 22 elderly patients were analyzed by Congo red staining for amyloid deposits. All samples contained amyloid, which was resistant to the potassium permanganate reaction. Tryptophan was present in all amyloid deposits. The amyloid failed to react with antiserums to amyloid fibril protein ASc1 or human prealbumin, proteins previous demonstrated in generalized senile cardiac amyloid. It also differed from age-related isolated atrial amyloid, which has been shown to lack tryptophan. Deposits did not react with antiserums specific for amyloid fibril proteins of the A lambda IV, A lambda VI, AA, or AEt types. These results indicate that senile aortic amyloid is distinct from amyloid present in primary and secondary amyloidosis and appears to represent a third form of cardiovascular amyloid associated with the aging process.     

Murine alveolar hydatidosis: a potential experimental model for the study of AA-amyloidosis

Histochemical and immunohistologic evidence has been presented to characterize the AA-type of amyloidosis in C57BL/6J (H-2b) mice infected i.p. with 50 cysts of Echinococcus multilocularis. Congo red-stained sections of kidneys and spleens from 4 weeks postinfected (p.i.) hydatid-mice were negative for the amyloid deposits. Heavy amyloid deposits, which in ultrathin sections of kidneys measured 8-12 nm in thickness, obliterated the perifollicular sinuses in spleens and glomerular capillaries in kidneys at 16 weeks p.i. Amyloid deposits were resistant to potassium permanganate treatment. They bind strongly to rabbit anti-mouse AA serum (RAA) as demonstrated by using peroxidase-anti-peroxidase technique. Preabsorption of RAA with azo-casein induced amyloid abolished completely the binding of RAA to mouse AA and hydatid-mouse deposits. Rabbit monospecific mouse antisera to heavy and light chains of Igs did not bind to amyloid deposits in hydatid-mice kidneys. Enumeration of spleen cells from the 16 weeks p.i. amyloidotic spleens showed a significant reduction in the total lymphocytes and T-cells. Overt accumulation of amyloid deposits in the spleen was associated with its disorganization, a significant reduction in T-cells and the depressed response of spleen cells to ConA and LPS mitogens. The relationship between proliferating alveolar hydatid cyst, intense inflammatory response, depressed cell mediated immunity and AA-type of amyloidosis is discussed in murine hydatidosis.     

Murine alveolar hydatidosis: a potential experimental model for the study of AA-amyloidosis.

Histochemical and immunohistologic evidence has been presented to characterize the AA-type of amyloidosis in C57BL/6J (H-2b) mice infected i.p. with 50 cysts of Echinococcus multilocularis. Congo red-stained sections of kidneys and spleens from 4 weeks postinfected (p.i.) hydatid-mice were negative for the amyloid deposits. Heavy amyloid deposits, which in ultrathin sections of kidneys measured 8-12 nm in thickness, obliterated the perifollicular sinuses in spleens and glomerular capillaries in kidneys at 16 weeks p.i. Amyloid deposits were resistant to potassium permanganate treatment. They bind strongly to rabbit anti-mouse AA serum (RAA) as demonstrated by using peroxidase-anti-peroxidase technique. Preabsorption of RAA with azo-casein induced amyloid abolished completely the binding of RAA to mouse AA and hydatid-mouse deposits. Rabbit monospecific mouse antisera to heavy and light chains of Igs did not bind to amyloid deposits in hydatid-mice kidneys. Enumeration of spleen cells from the 16 weeks p.i. amyloidotic spleens showed a significant reduction in the total lymphocytes and T-cells. Overt accumulation of amyloid deposits in the spleen was associated with its disorganization, a significant reduction in T-cells and the depressed response of spleen cells to ConA and LPS mitogens. The relationship between proliferating alveolar hydatid cyst, intense inflammatory response, depressed cell mediated immunity and AA-type of amyloidosis is discussed in murine hydatidosis.     

Histopathological diagnosis of amyloidosis

For the diagnosis of amyloidosis, histological evidence of amyloid deposition is essential. Histologically, an amyloid deposit is stained orange red with Congo red and shows green birefringence under polarized light. When amyloidosis is clinically suspected, endoscopic biopsy of the stomach, duodenum or colon, or aspiration biopsy of abdominal fat is usually performed. If clinicians suspect amyloidosis, they should advise pathologists. Identification of the chemical type of amyloid is necessary with respect to treatment and prognosis. Immunohistochemical examination of amyloid in formalin-fixed, paraffin-embedded sections is simple to perform in most pathological laboratories. In Japan, almost all cases of systemic amyloidosis are classified as AL, AA, ATTR or Abeta2M amyloidosis, so the use of anti-immunoglobulin light chain, anti-amyloid A, anti-transthyretin and anti-beta2 microglobulin antibody is recommended for the classification of systemic amyloidosis. Formic acid pretreatment, which is often used for immunohistochemical detection of amyloidosis, is useful and easy for antigen retrieval. Amyloid deposits of AL amyloidosis are sometimes not immunostained well with commercial anti-immunoglobulin light chain antibody. Previously, we generated polyclonal antibodies against synthetic peptides corresponding to positions 118-134 of immunoglobulin lambda light chain and positions 116-133 of immunoglobulin kappa light chain. These antibodies are very useful for detecting AL amyloidosis because they react with amyloid deposits on formalin-fixed, paraffin-embedded specimens in almost all AL amyloidosis cases. Exact diagnosis and typing of amyloidosis are necessary for therapy.     

Amyloid A gastrointestinal amyloidosis associated with idiopathic retroperitoneal fibrosis. Report of a rare autopsy case and review of the literature

We report a rare autopsy case of secondary gastrointestinal amyloid A (AA) amyloidosis associated with idiopathic retroperitoneal fibrosis (IRF) in a 67-year-old woman. Masses were identified around the aorta and inferior vena cava in her abdomen. Histologically, plasma cell infiltration was observed within fibrotic areas. Because no specific cause for the inflammatory mass was apparent, we diagnosed it as IRF. Steroid therapy, which usually reduces IRF masses, proved ineffective, and malabsorption syndrome developed 4 years later. On autopsy, amyloid protein was present systemically in the vascular walls of several organs, and deposition was highest in the gastrointestinal mucosa. Amyloid protein was identified as AA type, strongly suggesting that the amyloidosis was secondarily induced by IRF. To our knowledge, only 2 other cases of IRF-associated amyloidosis have been reported. Two of the 3 patients of these cases were women who showed resistance to steroid therapy.