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.     

Hepatic amyloidosis: morphologic differences between systemic AL and AA types

The liver is almost universally involved in systemic amyloidosis. Patterns of topographic distribution of amyloid within the liver lobule have been recognized, but the reliability of using these for classification of amyloid type is in question. We examined 286 livers from cases of systemic amyloidosis obtained from autopsies at Los Angeles County-University of Southern California Medical Center, classifying them as AL or AA type by means of the potassium permanganate Congo red-staining method along with a specific anti-AA antiserum. Prior publications have asserted that deposition of secondary (AA) amyloidosis is limited to the vessels in the portal tract, constituting a "vascular" pattern, and that in primary (AL) amyloidosis the deposits exhibit a "sinusoidal" pattern in that they are seen along hepatic sinusoids as well as in portal vessels. We confirmed that AL amyloid involves the portal vessels as frequently as AA amyloid and that deposition occurred significantly more frequently in the portal stroma, the central vein, and the "sinusoidal" areas. However, we also found a "sinusoidal" pattern in 29 of 78 cases of secondary (AA) amyloidosis; in 14 of these, more than half of the sinusoidal spaces were replaced by amyloid deposits. We also noted that in 23 of the 29 AA amyloidosis cases with "sinusoidal" involvement, a "sago" pattern of distribution of amyloid in the spleen was present. No consistent association of a specific chronic inflammatory disease with "sago" spleen and "sinusoidal" deposits could be documented. We conclude that topographic distribution of amyloid within the liver lobule is not a reliable method of distinguishing AA from AL amyloidosis and that specific staining methods must be used if the physician is to be able to attempt modern therapeutic modalities.     

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.     

Amyloiddiagnostik bei rheumatischen Erkrankungen

Amyloid is a pathological protein deposit in tissue which has a red eosin color when the slice preparation is stained with traditional hematoxylin and eosin and after Congo red staining under polarized light exhibits a characteristic apple-green polarization color. Over 26 different autologous physiological proteins have been described that can form amyloid. In surgical pathology, immunoglobulin light chain-associated AL amyloidosis is the most frequent generally occurring amyloidosis, followed by hereditary and nonhereditary ATTR amyloidosis and AA amyloidosis. AA amyloidosis mostly develops subsequent to chronic infectious or inflammatory underlying disease and can represent a potentially life threatening complication. The spectrum of causes for AA amyloidosis has changed in the past few decades and is now determined by chronic rheumatic diseases and hereditary periodic fever syndromes. Early diagnosis of an amyloidosis and its correct classification continue to pose a great challenge. Precise classification of the amyloid and amyloidosis is essential for prognosis assessment and treatment planning. In addition to anti-inflammatory management of AA amyloidosis, specific treatment strategies may possibly become available in the future.     

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.     

An investigation of the protein components of amyloid using immunoperoxidase and permanganate methods on tissue sections

Amyloid deposits in tissue from 8 patients with generalized primary amyloidosis, 11 patients with generalized secondary amyloidosis, 11 nasopharyngeal carcinomas, 11 basal cell carcinomas, 4 islet cell tumours, 4 medullary carcinomas of the thyroid and 9 cases of lichen amyloidosis were studied using the indirect immunoperoxidase and peroxidase-antiperoxidase methods with specific antisera against Amyloid A (AA) protein and human immunoglobulin lambda and kappa light chains. The permanganate method of Wright was also applied to tissue sections. Positive staining for AA protein was observed only in secondary amyloidosis. There was excellent correlation between AA positivity and permanganate sensitivity. Positivity for immunoglobulin light chains was not observed in secondary amyloidosis but was noted in 5 (63%) cases of primary amyloidosis and 18-27% of intratumour amyloidosis. Lichen amyloidosis did not stain for AA protein or light chains. It is shown that assessment of the permanganate reaction and AA positivity of amyloid deposits can reliably differentiate secondary from primary amyloidosis and may contribute significantly to selection of patients for appropriate therapy.     

Abdominal fat tissue aspirate in human amyloidosis: light, electron, and immunofluorescence microscopic studies

Abdominal fat tissue aspirates from 12 patients with biopsy-proved amyloidosis were investigated by different morphologic techniques. By light microscopy, after staining of the fat tissue aspirates with Congo red and examination with a polarizing microscope, positive results were obtained in nine patients with amyloidosis, two of the three with primary (AL) amyloidosis and seven of the nine with secondary (AA) amyloidosis. By indirect immunofluorescence, using AA antiserum, positive results were obtained in five of the nine cases of AA amyloidosis (aspirates from these five patients were positive on Congo red staining). By electron microscopy, amyloid fibrils were observed in five cases of amyloidosis (two of the AL and three of the AA type, all positive on Congo red staining). Although amyloid was demonstrated less frequently by immunofluorescence and electron microscopy, perhaps because of the small numbers of fat particles examined, it seems that, with Congo red staining, abdominal fat tissue aspiration is a simple and sensitive method for the diagnosis of amyloidosis. Immunofluorescence studies allow discrimination between the different types of amyloidosis. The method could be used in patients in whom other types of tissue biopsy are not recommended because of risks of bleeding or other problems.     

Fibronectin and basement membrane components in renal amyloid deposits in patients with primary and secondary amyloidosis.

Kidney biopsies from one patient with primary (AL) and three with secondary (AA) amyloidosis were used for an ultrastructural study of the collocalization of basement membrane proteins and the extracellular matrix protein fibronectin within amyloid deposits. Antibodies against amyloid P component, laminin, and heparan sulphate proteoglycan core protein all reacted with the basement membranes and the amyloid depositions in AA and AL amyloidosis. Monoclonal and polyclonal antibodies against collagen type IV reacted only with the basement membranes. Anti-fibronectin reaction was found in association with the basement membranes in all four cases, while labelling of amyloid depositions was found only in one of the AA amyloid cases and in the AL amyloid depositions. It is concluded that basement membrane components may be of importance for the formation of amyloid fibrils.     

Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain kappaIII subtype in a small formalin fixed, paraffin wax embedded tissue sample

BACKGROUND: Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins. AIMS: To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type. METHODS: Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing.RESULTS: Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain kappaIII subtype (AL-kappaIII) (16, 8, and 3 kDa). CONCLUSIONS: This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive.     

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).     

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     

Primary amyloidosis A. Immunohistochemical and biochemical characterization.

Primary "idiopathic" amyloidosis is usually related to immunoglobulin light chain (AL) associated with immunocytic dyscrasias, while secondary "reactive" amyloidosis (AA) is related to serum amyloid A protein (SAA) and typically occurs with chronic inflammation, malignancy, or familial Mediterranean fever. In the present study, amyloid fibril protein extracted from frozen and paraffin-embedded tissue from a patient (CAR) with primary systemic amyloidosis proved to be AA protein by immunohistochemical, immunochemical, and amino terminal sequence. Extracts from both frozen and formalin-fixed paraffin-embedded kidney and spleen yielded similar monomers and dimers of the AA protein. The additional high-molecular-weight bands and a distinct 12,000-dalton fragment in the amyloid protein extracted from the formalin-fixed paraffin-embedded lung suggest that different processing of proteins, ie, by polymerization and/or degradation, may occur in different organs.     

Gastrointestinal amyloid deposition in AL (primary or myeloma-associated) and AA (secondary) amyloidosis: Diagnostic value of gastric biopsy

Gastrointestinal amyloid deposition was investigated in 21 autopsy cases of nonhereditary systemic amyloidosis, 18 of the AL (primary or myeloma-associated) type and three of the AA (secondary) type. Vascular deposition of amyloid, most apparent in the submucosa, was found in all cases. Parenchymal deposition was observed mainly in the muscularis mucosae and muscularis externa in the AL type, and in the lamina propria mucosae in the AA type. Comparison of amyloid deposition in the stomach and rectum revealed no differences for the AA type. In the AL type, however, deposition in the lamina propria mucosae and muscularis mucosae was more frequent and marked in the wall of the stomach than in the rectum. Thus, gastric biopsy would be more valuable than rectal biopsy in the diagnosis of AL amyloidosis.     

Identification and Characterization of Different Amyloid Fibril Proteins in Tissue Sections

Antisera specific for 4 different classes of amyloid fibril proteins, amyloid protein AA and the immunoglobulin light-chain amyloid proteins A_λI, A_λIV, and A_λV, were used to identify these proteins directly in tissue sections from 25 patients with amyloidosis. The specificity of these reactions was established by blocking experiments with purified amyloid fibril proteins and Bence Jones proteins of known variable subgroups. Protein AA was detected in 17 patients, including all 13 with secondary amyloidosis, 2 with primary amyloidosis and 2 with Waldenström's macroglobulinemia. Immunoglobulin light-chain proteins A_λ I, A_λIV, and A_λ V were found in 3, 1, and 2 patients, respectively, all of whom had primary or myeloma/macroglobulinemia-associated amyloidosis. Antiserum specific for the amyloid-related serum protein SAA reacted with the same tissues as anti-AA and had the same pattern of staining in tissue sections.     

Primary localized amyloidosis of the bladder with a monoclonal plasma cell infiltrate

A case of primary tumour-like amyloidosis of the urinary bladder is described. The plasma cell infiltrate around the amyloid deposition revealed monoclonality by both immunoperoxidase and immunofluorescence methods and the amyloid gave a staining reaction consistent with AL type. A local tissue based immunocyte dyscrasia appears to be responsible for the tumour-like amyloid deposition.     

Hepatic amyloidosis. A histopathologic analysis of primary (AL) and secondary (AA) forms.

The liver is a major site of amyloid deposition. The spectrum of histopathologic changes in the liver was studied in 38 patients with systemic amyloidosis (25 with primary or myeloma-associated amyloidosis [AL] and 13 with secondary, reactive [AA] amyloidosis). Overall architectural distortion, alterations of portal triads, as well as predilection for topographic deposition in the parenchyma and/or blood vessel walls were noted. Significant histopathologic differences in AL or AA amyloid liver involvement included 1) portal fibrosis, seen in 7 of 25 (28%) AL patients and 8 of 13 (62%) AA patients (P = 0.05), 2) parenchymal amyloid deposition in 25 of 25 (100%) AL amyloid and 10 of 13 (77%) AA amyloid patients (P = 0.04), and 3) vascular amyloid deposition found in 17 of 25 (68%) with AL amyloid and 13 of 13 (100%) patients with AA amyloid (P = 0.02). These data vary from the widely held concept that deposition of amyloid is predominantly vascular in the AL form and parenchymal in amyloid AA. Clearly, however, in individual cases significant overlap occurred, and characterization of amyloid types based on morphologic distribution of amyloid deposits may be possible in only a minority of cases. In most cases, differentiation of amyloid AL and amyloid AA forms requires clinical, histochemical, immunochemical, and sometimes more elaborate laboratory amino acid sequence studies for accurate identification.     

Primary localized amyloidosis of the genitourinary tract: immunohistochemical study on eleven cases

Eleven cases of primary localized amyloidosis of the genitourinary tract were studied using the unlabeled immunoperoxidase method in combination with specific antisera against different chemical types of amyloid fibril proteins (AA,A lambda, and A kappa). Nine of these were shown to have A lambda antigenic fibril determinants, suggesting that the fibrils of most, if not all, of these cases are of immunocytic origin.     

Splenic amyloidosis: correlations between chemical types of amyloid protein and morphological features

Sixty-one autopsy cases of splenic amyloidosis were reviewed to assess the relationship between the morphological patterns and chemical types of amyloid protein. On the basis of immunohistochemical reactions of amyloid protein, the cases were classified into 34 cases of AA and 27 of AL amyloidosis. Amyloid deposition in the spleen was divided into three major sites: the red pulp, the white pulp, and blood vessels. Red pulp involvement by amyloid was noted in 52% of the AL cases but in none of the AA cases. White pulp amyloid deposition was found in 70% of the AL and 35% of the AA cases. This difference was statistically significant (P less than 0.001). On the other hand, vascular deposition of amyloid was invariably noted in all cases with AA or AL amyloidosis, affecting the AA cases rather severely. These results strongly suggest that the widely held concept of deposition of amyloid as predominantly vascular in AL amyloidosis and parenchymal in AA amyloidosis requires revision. Our findings indicate that parenchymal, especially the red pulp, involvement is a consistent feature of AL amyloidosis, whereas vascular involvement is a finding common to both types of systemic amyloidosis.     

Diagnosis and treatment in systemic amyloidosis

Systemic amyloidosis is characterized by the involvement of multiple organs and the presence of an amyloid precursor protein in serum. This disorder is classified into four major forms: immunoglobulin light chain-derived (AL), reactive AA, dialysis-related (beta2M) and hereditary transthyretin (ATTR) type. Heart, kidney, gastrointestinal tract and peripheral nerves are commonly affected by amyloid deposition in systemic amyloidosis and histopathological demonstration of amyloid deposits on any of affected organs is the first step leading to the diagnosis of this disease. Immunohistochemical analysis of amyloid protein on tissue amyloid deposits is necessary to make classification of the disease and DNA testing is also useful in a hereditary form. Amyloidosis had been considered to be an incurable disease but during the past one decade several therapeutic approaches have been employed for the amyloidosis patients with diverse pathogenetic backgrounds: intravenous large dose of melphalan accompanied by autologous peripheral blood stem cell transplantation for AL amyloidosis and liver transplantation for hereditary ATTR type amyloidosis. As a result some amyloidosis patients have been rescued and are now enjoying their own social lives. It is likely that recent advance on the research of amyloidosis has changed the concept of this disease.