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 distinction between amyloidosis and fibrillar glomerulopathy.

Six patients with glomerulonephritis and glomerular proteinaceous deposits constituted by fibrillar ultrastructures similar to those of amyloid but lacking the Congo red tinctorial affinity characterizing amyloid were studied. Clinically, these patients had proteinuria and hematuria; in addition, three patients had hypertension and one renal failure. Protein deposits in their kidney biopsy sections were evaluated by immunofluorescence, immunoperoxidase, and immunoelectron microscopic (protein A-gold) techniques, using antibodies against IgG, IgA, IgM, C3, C1q, fibrinogen, immunoglobulin kappa and lambda light chains, and against amyloid fibril proteins of different types, including AA, A lambda, A kappa, and AF. By immunofluorescence and immunoperoxidase, in all cases the deposits stained intensely with antibodies against IgG, C3, and kappa and lambda light chains; one case also showed C1q immunoreactivity. By contrast, none stained with antibodies against various amyloid fibril proteins. Immunoelectron microscopic findings corroborated this data, indicating that the nonamyloid fibrillar deposits studied are antigenically distinct from known amyloid deposits and that they contain IgG-derived material.     

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.     

Nodular glomerulopathy associated with nonamyloidotic kappa light chain deposits and excess immunoglobulin light chain synthesis

A nodular glomerulopathy characterized by mesangial deposits of monoclonal kappa light chains was detected by immunofluorescence in a renal biopsy from a patient with proteinuria and hypertension. These nodules lacked the tinctorial and morphologic features of amyloid. Ultrastructurally, the nodules contained electron-dense granular deposits as well as fibrils in parallel arrangement. The fibrils measured 110-140 A in diameter. They were consistent in size with amyloid fibrils. However, they differed in lacking the randomly oriented network of typical amyloid fibrils and more closely resembled fibrils intrinsic to mesangial matrix. The patient had no bone marrow or X-ray evidence of myeloma and no evidence of free monoclonal light chains in serum or concentrated urines. Biosynthetic studies of the patient's bone marrow cells demonstrated unbalanced immunoglobulin synthesis with excess production of monoclonal kappa light chains. These observations suggest that the observed glomerulopathy results from direct deposition of monoclonal light chains. Deposits with kappa light chain determinants have been found in 7 other patients with similar nodular glomerulopathies, 4 of whom had diagnosed clinical myeloma. The lesion of nonamyloidotic nodular glomerulopathy previously described in 19 patients, nor examined by immunopathologic techniques or not shown to contain light chain determinants, may have a similar pathogenesis.     

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.     

Classification of amyloid deposits in diagnostic cardiac specimens by immunofluorescence

BackgroundAt least 12 distinct forms of amyloidosis are known to involve the heart or great vessels. Patient treatment regimens require proper subtyping of amyloid deposits in small diagnostic cardiac specimens. A growing lack of confidence in immunohistochemical staining for subtyping amyloid has arisen primarily as a result of studies utilizing immunoperoxidase staining of formalin-fixed paraffin-embedded tissue. Immunofluorescence staining on fresh frozen tissue is generally considered superior to immunoperoxidase staining for subtyping amyloid; however, this technique has not previously been reported in a series of cardiac specimens.MethodsAmyloid deposits were subtyped in 17 cardiac specimens and 23 renal specimens using an immunofluorescence panel.ResultsAmyloid deposits were successfully subtyped as AL, AH, or AA amyloid by immunofluorescence in 82% of cardiac specimens and 87% of renal specimens. In all cases, the amyloid classification was in good agreement with available clinical and laboratory assessments. A cross-study analysis of 163 cases of AL amyloidosis reveals probable systemic misdiagnosis of cardiac AL amyloidosis by the immunoperoxidase technique, but not by the immunofluorescence technique.ConclusionsAmyloid deposits can be reliably subtyped in small diagnostic cardiac specimens using immunofluorescence. The practical aspects of implementing an immunofluorescence approach are compared with those of other approaches for subtyping amyloid in the clinical setting.     

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.     

Kappa light chain glomerulosclerosis in multiple myeloma

Kappa light chains were demonstrated by immunofluorescence microscopy in the renal basement membranes of 6 of 10 patients with multiple myeloma subjected to kidney biopsy. Nodular glomerulosclerosis was present in 2 of these patients, but the remainder showed only minor light microscopic abnormalities. Deposition of kappa light chains in renal basement membranes may be a frequent cause of kidney disease in multiple myeloma. This deposition may be detected with electron microscopy by the demonstration of granular dense transformation of basement membranes. Occasionally there is systemic vascular deposition of kappa light chains which may be difficult to distinguish from amyloid but lacks a fibrillar character. The tissue effects of light chains in multiple myeloma seem likely to be more extensive than their deposition in renal tubules. There appears to be a greater tendency for kappa light chains to accumulate in renal and systemic basement membranes, but the mechanisms governing this deposition are unknown.     

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.     

The classification of amyloid deposits in clinicopathological practice

A series of 104 biopsy cases with histopathological proof of amyloid, submitted to our department of pathology over the last 19 years, were re-examined. The survey investigated the medical indication for surgery, the origin and quality of the biopsy and the clinical information as documented on the request form for histopathological examination and in hospital records. Amyloid deposits were classified using antisera directed against five major amyloid fibril proteins, i.e. AA, ATTR, Aλ, Aκ and Aβ2M and optimal conditions were sought for the reliable and early characterization of amyloid disease in clinicopathological practice. This survey revealed that 98% of the biopsy cases already suffered from a disease which was either a cause or a result of amyloidosis. In only 2% of the biopsy cases was amyloidosis detected without any clinical indication. Immunohistochemical classification of the amyloid deposits and comparison with hospital records demonstrated diagnostic pitfalls such as immunostaining of amyloid by two or more antibodies recognizing different fibril proteins, and disagreement between immunohistochemical typing of amyloid and the initial clinical diagnosis. Based on these observations we assume that the characterization of amyloid disease and its biological significance is impossible in clinicopathological practice without clinical information or without immunohistochemical classification of the fibril protein in biopsy specimens. Different aspects of histopathological detection of AA- and AL-amyloidosis are discussed.     

Immunohistological characterisation of amyloid deposits in renal biopsy specimens.

The amyloid deposits in 21 renal biopsy specimens were subjected to a detailed immunohistochemical analysis using a panel of antibodies against recognised constituents of tissue amyloid. This was a retrospective study of material originally submitted during the investigation of various renal abnormalities and studied by a routine protocol including histochemistry, electron microscopy, and immunofluorescence. The presence of an amyloid was confirmed in all 21 cases. Seventeen cases contained P component and either amyloid A (AA) (11 cases) or an immunoglobulin light chain associated amyloid (six cases). Four cases contained amyloid material with unusual immunohistochemical findings; one case had AA and P-component (PC) in the interstitium, one case had lambda light chain and beta-2 microglobulin, one case had kappa light chain and Clq, and one case had lambda light chains only. It was possible, therefore, to identify precisely the amyloid constituents and thereby "type" the amyloid by immunohistochemical means. The availability of the antibodies used and their application using these techniques could simplify the confirmation of clinically suspected amyloidosis.     

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.     

Amyloid in endomyocardial biopsies

The prognosis of cardiac amyloidosis depends on the nature and origin of the amyloid protein deposited. However, little is known about the prevalence and origin of amyloid in heart muscle biopsies. We therefore examined retrospectively the distribution and origin of amyloid in a consecutive series of endomyocardial biopsies. Endomyocardial biopsies with verified presence of amyloid from 101 patients were included. Amyloid was classified immunohistochemically in each of them. Our collective comprised 63 men and 38 women, with a mean age of 66 years (range 37–85 years). Cardiac amyloidosis was the most common of the AL (54 patients) or ATTR type (42 patients). In five individuals, amyloid remained unclassified. AL amyloidosis was subdivided into ALλ (45 patients) and ALκ amyloid (nine patients). AA amyloid was not found in any individual. The amount of amyloid was higher in AL than in ATTR amyloidosis. Genomic DNA was extracted and examined by DNA sequencing in 19 patients with ATTR amyloidosis. Five (26%) individuals carried TTR mutations (p.Val20Ile, p.Val30Met (twice), p.Asp39Val and p.Glu54Asp) and were classified as suffering from hereditary ATTR amyloidosis. Amyloid in endomyocardial biopsies is most commonly of immunoglobulin light chain origin, followed by non-hereditary and hereditary-type ATTR amyloid.     

Induction in mice of human light-chain-associated amyloidosis.

Primary (idiopathic) or multiple myeloma-associated amyloidosis is characterized by the deposition in tissue of monoclonal light chains or light-chain fragments (AL amyloidosis). In contrast to other types of amyloidosis, information regarding the pathogenesis of light-chain-related amyloid has heretofore been limited due to the lack of a suitable in vivo model. The authors report the successful experimental induction of human AL amyloid deposits. The repeated injection into mice of Bence Jones proteins obtained from two patients with AL amyloidosis produced the histopathologic lesions characteristic of this disease. Partial dehydration of animals before protein injection resulted in the acceleration of amyloid formation. The human proteins were deposited as amyloid within the mouse renal blood vessel walls and parenchymal tissue, as well as in other organs. The deposits were Congo red-positive, exhibited green birefringence, and had a fibrillar ultrastructure. As evidenced immunohistochemically, the experimentally induced amyloid deposits consisted of the injected human light chains, and in addition, contained mouse amyloid P component (AP); mouse immunoglobulin (Ig) or inflammatory-associated amyloid A protein was not detected. Extraction and characterization of the amyloid deposits found within the mouse kidney revealed the presence of a predominantly intact human light polypeptide chain. Mice injected in identical manner with a non-amyloid-associated Bence Jones protein had no or only rare amyloid deposits. The experimental mouse model provides a means to ascertain the amyloidogenic potential of human monoclonal light chains and to study further the pathogenesis of AL amyloidosis.     

Immunological characterization of amyloid fibrils in tissue sections

The labelling of rabbit antiserum against purified and alkaline-degraded amyloid fibrils with fluorescein isothiocyanate (FITC), has enabled the detection of amyloid deposits in various tissue sections by direct immunofluorescence. There was antigenic identity between amyloid from different organs of the same patient. In addition, indirect immunofluorescence revealed individual antigenic specificity and cross-reactivity among amyloid from different individuals. A close relationship between deposits of amyloid, immunoglobulins and complement was observed when using FITC-and rhodamine-labelled antisera against various human plasma components.     

Nonamyloidotic monoclonal immunoglobulin deposits lack amyloid P component

Deposits in tissues from 13 patients with amyloid, 8 with monoclonal light chain or light and heavy chain deposition disease, and 2 with both amyloid and nonamyloidotic light chain deposits of the same isotype were examined in parallel for the presence of amyloid P component by immunofluorescence and/or immunoperoxidase methods. Amyloid P component was detected in the amyloid but not the nonamyloid deposits, even in the 2 individuals in whom both types of deposits were present, indicating a specific relationship between the amyloid P component and the amyloid fibrils.     

Localized primary amyloid tumor of the thyroid developing in the course of Hashimoto's thyroiditis.

A case of localized primary amyloid tumor of the thyroid gland developing in the course of Hashimoto's thyroiditis was studied using histochemistry, immunohistochemistry and electron microscopy. The patient was diagnosed as having Hashimoto's thyroiditis by histological examination of the thyroid and by the presence of a high titer of serum thyroglobulin and thyroid microsomal antibodies. In addition, the thyroid gland exhibited multiple nodular deposits of amyloid which were resistant to prior incubation with potassium permanganate. The amyloid deposits were surrounded by numerous histiocytes and multinucleated giant cells which contained small amyloid droplets in their cytoplasm. However, no amyloid deposits were observed in the walls of blood vessels. Immunohistochemistry showed that the amyloid was strongly positive for amyloid P component, IgG and kappa light chains. Ultrastructurally, the amyloid was composed of straight fibrils with a diameter of 7 to 10 nm. Histiocytes extended slender cytoplasmic processes in a radial fashion into amyloid fibrils, which exhibited a highly organized star-like pattern. This was considered to be an extremely rare case of localized primary amyloidosis of the thyroid, in which IgG, especially kappa light chains (AL), was present as a precursor protein.     

Individual antigenic specificity and cross-reactions among amyloid preparations from different individuals

Amyloid fibrils were isolated from eleven amyloid-laden organs of six patients. By alkaline degradation, soluble units were obtained which gave antibody formation in rabbits. Gel precipitation and haemagglutination inhibition were used to characterize antigens of the amyloid. Evidence was obtained that amyloids from different organs of the same individual were identical in the antigenicity. In contrast, amyloids from different individuals each showed unique individual specificity. Besides this, antigenic cross-reactions were noted between the amyloid preparations. Finally, evidence for antigenic cross-reactivity between certain amyloid preparations and immunoglobulin light chains was obtained.     

Localized Primary Amyloid Tumor of the Thyroid Developing in the Course of Hashimoto's Thyroiditis

A case of localized primary amyloid tumor of the thyroid gland developing in the course of Hashimoto's thyroiditis was studied using histochemistry, immunohistochemistry and electron microscopy. The patient was diagnosed as having Hashimoto's thyroiditis by histological examination of the thyroid and by the presence of a high titer of serum thyroglobulin and thyroid microsomal antibodies. In addition, the thyroid gland exhibited multiple nodular deposits of amyloid which were resistant to prior incubation with potassium permanganate. The amyloid deposits were surrounded by numerous histiocytes and multinucleated giant cells which contained small amyloid droplets in their cytoplasm. However, no amyloid deposits were observed in the walls of blood vessels. lmmunohistochemistry showed that the amyloid was strongly positive for amyloid P component, IgG and kappa light chains. Ultrastructurally, the amyloid was composed of straight fibrils with a diameter of 7 to 10 nm. Histiocytes extended slender cytoplasmic processes in a radial fashion into amyloid fibrils, which exhibited a highly organized star-like pattern. This was considered to be an extremely rare case of localized primary amyloidosis of the thyroid, in which IgG, especially kappa light chains (AL), was present as a precursor protein. Acta Pathol Jpn 42: 210–216. 1992.     

Portal amyloid: novel amyloid deposits in gastrointestinal veins?

OBJECTIVE: To specify uncharacterized amyloid deposits in gastrointestinal vessels of the elderly. MATERIALS AND METHODS: The gastrointestinal tracts from 110 consecutive autopsies of individuals aged 85 years and older were examined for amyloid using Congo red staining. Immunohistochemical classification of the amyloid deposits was conducted using antisera directed against amyloid A, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, apolipoprotein C-I, lysozyme, lambda and kappa light chain amyloid fibril proteins, transthyretin, beta2-microglobulin, and amyloid P component. Electron microscopic examination assessed the ultrastructural features. RESULTS: Thirty-eight (35%) of the 110 cases had gastrointestinal amyloid deposits. In 17 cases the amyloid fibril proteins were defined immunohistochemically. In five cases (5%) the amyloid could not be classified because amyloid deposits were not present in the deeper serial sections used for immunohistochemistry. In 13 cases (11%) the vascular amyloid deposits could not be characterized because they did not demonstrate immunoreactivity with any of a panel of antibodies specific for the fibril proteins of all major extracerebral amyloids. In three individual cases, the vascular amyloid deposits showed variable immunoreactivity, with deposits being negative in some vessels. The immunohistochemically nonreactive vascular amyloid in these 16 cases had several consistent features: it affected only vessels of the small and large intestine, it was limited to mesenteric veins, it consisted of small dot- or comma-like deposits located in close proximity to fragmented elastic fibers, and it demonstrated inconsistent immunostaining for amyloid P component. CONCLUSIONS: The similar morphologic characteristics of nonreactive gastrointestinal amyloid deposits, which we have designated "portal amyloid," suggest a common origin. Determination of whether portal amyloid represents a new type of amyloid will require chemical analysis.