Topo-optical visualization reactions of carbohydrate-containing amyloid deposits in the respiratory tract

Staining with Congo red according to is the most commonly used method for the demonstration of amyloid, but structures other than amyloid can give false-positive results. To overcome this problem, introduced an aqueous Congo red staining with gum arabic as the mounting medium, which we have used in this and previous publications. Most histochemical studies on amyloid deposits to date have concentrated on conventional methods including staining with thioflavine, sirius red, alcian blue, methyl and crystal violet. In this study, we used topo-optical reactions with thiazine dyes on both the light and polarization microscopic level to establish the structure, distribution and location of carbohydrate components that occur within amyloid deposits, especially in the respiratory tract. Topo-optical staining reactions for the qualitative analysis of carbohydrate components in amyloid deposits included (1) reactions that identify the carbohydrate residues, (2) reactions that detect sialic acids and, (3) methods that visualize glycosaminoglycans. In conclusion, a comparison of consecutive serial sections stained with Congo red, aldehyde bisulfite toluidine blue reaction, sialic acid-specific topo-optical reaction, toluidine blue topo-optical reaction and chemically intensified basophilic reaction showed correlative staining patterns and anisotropic effects, corresponding to a close pathomorphological relationship between amyloid fibrils, periodate reactive carbohydrates, including sialic acids, and glycosaminoglycans.     

Histochemical analysis of senile plaque amyloid and amyloid angiopathy

Summary Histochemical methods were used to obtain information on the chemical constituents of brain amyloid in senile dementia of the Alzheimer type. The staining properties of brain amyloid (senile plaque and amyloid angiopathy) were compared with those of extraneural amyloidosis and endocrine amyloid. We found no histochemical differences between amyloid in senile plaques and in amyloid angiopathy. The content of aromatic amino acids was higher in amyloid of plaques and in amyloid angiopathy than in endocrine amyloid. Furthermore, we found persistent birefringence and affinity of brain amyloid for Congo red after exposure to potassium permanganate, suggesting that AA amyloid is not a major constituent of cerebral amyloid.     

Amyloid deposits in pituitary adenomas. Differentiation of two types

Two different types of amyloid deposits in pituitary adenomas are described herein. The deposits of the first type are stellate and perivascular masses, with fibrillary inclusions occurring in the intercellular space. No immunoreactive cytokeratin can be detected in the deposits, and the masses are secreted by the adenoma cells. A second, rarer type is characterized by spheroids (40 to 1500 micron in diameter) in which immunoreactive cytokeratin fibrils are present. The fibrils originate in the adenoma cells adjacent to the deposits. The amyloid nature of both types of deposits can be proved by intense red staining with Congo red, green dichroism, and positive immunostaining with antibodies raised against serum amyloid P component. Adenomas with amyloid spheroids are very rare, and most have been proved or suspected prolactin-producing adenomas.     

Unusual renal deposit in mixed connective tissue disease.

We describe a patient with clinical signs of mixed connective tissue disease who developed nephrotic syndrome. The kidney biopsy revealed glomerular and vascular deposits that stained positive with Congo red and showed a green birefringence. The Congo red positivity became negative in sections treated with potassium permanganate. Peroxidase-antiperoxidase staining with anti-AA antibodies was strongly positive. Ultrastructurally, although the deposits were similar to amyloid, they were about twice the size of amyloid fibrils. To our knowledge, the deposition of an amyloidlike material that shows the histochemical and immunohistochemical features of amyloid AA but is lacking the distinctive ultrastructural characteristics of amyloid fibrils has not been described.     

Prevalence of amyloid and positive Congo red frequency among routine surgical specimens

Amyloidosis is characterized by an extracellular tissue deposition of one of a family of biochemical proteins that are abnormally folded. The deposits are often subtle, and can be missed on routine hematoxylin and eosin (H&E)-stained slides. Current literature does not offer an expected prevalence rate of amyloid or frequency of Congo red positivity among routine surgical pathology specimens in a referral bias-free setting. The objective of this study was to determine these parameters at a large community hospital. The pathology database was searched for all surgical pathology and autopsy cases diagnosed with amyloidosis from 2001–2013. All cases were reviewed and clinical parameters were recorded. Based on H&E interpretation, Congo red was performed on 218 cases. Of these, 36% confirmed positive birefringence. The prevalence of amyloid among routinely submitted pathology specimens was calculated as 0.027% over the 13-year interval. Amyloid was detected in less than 1% of routine surgical specimens. When suspicious H&E findings prompted Congo red staining, amyloidosis was confirmed about a third of the time over the 13-year period. Establishing an optimal rate of Congo red utilization may provide a standard measurement needed to ensure high amyloid detection rates among pathologists at the community level.     

Multinodular deposition of AA-type amyloid localized in the adrenal glands of an old man.

Multinodular amyloid deposits localized in non-neoplastic adrenal glands were found incidentally at autopsy in an 83-year-old Japanese man. Clinically, the patient lacked evident deficiency of adrenal hormones. The nodules of the stromal amyloid deposits were scattered in the adrenal cortex, where the parenchymal cells were compressed and atrophic. The deposits were confirmed to be amyloid by Congo red staining and polarization microscopy. Amyloid fibrils were also demonstrated in the deposits by electron microscopy. The amyloid deposits were permanganate-sensitive and showed immunohistochemical staining for serum amyloid P component and serum amyloid A protein (SAA), implying that they were AA amyloid. There have been no reports describing localized amyloid deposits of the AA type in non-neoplastic adrenal glands. The pathogenesis and clinical significance of the amyloid deposition in the present case remain only speculative.     

Multinodular Deposition of AA-type Amyloid Localized in the Adrenal Glands of an Old Man

Multinodular amyloid deposits localized in non neoplastic adrenal glands were found incidentally at autopsy in an 83-year-old Japanese man. Clinically, the patient lacked evident deficiency of adrenal hormones. The nodules of the stromal amyloid deposits were scattered in the adrenal cortex, where the parenchymal cells were compressed and atrophic. The deposits were confirmed to be amyloid by Congo red staining and polarization microscopy. Amyloid fibrils were also demonstrated in the deposits by electron microscopy. The amyloid deposits were permanganate-sensitive and showed immunohistochemical staining for serum amyloid P component and serum amyloid A protein (SAA), implying that they were AA amyloid. There have been no reports describing localized amyloid deposits of the AA type in non neoplastic adrenal glands. The patho-genesis and clinical significance of the amyloid deposition in the present case remain only speculative. Acta Pathol Jpn 42: 893–896, 1992.     

High prevalence of wild-type transthyretin deposition in patients with idiopathic carpal tunnel syndrome: a common cause of carpal tunnel syndrome in the elderly

Carpal tunnel syndrome is the most common type of entrapment neuropathy. However, the cause of carpal tunnel syndrome remains unclear in most cases. Senile systemic amyloidosis, induced by wild-type transthyretin deposition, is a prevalent aging-related disorder and often accompanied by carpal tunnel syndrome. In this study, we measured the frequency of unrecognized wild-type transthyretin deposition in patients with idiopathic carpal tunnel syndrome. One hundred twenty-three patients with carpal tunnel syndrome, including 100 idiopathic patients, treated by carpal tunnel release surgery were analyzed. Tenosynovial tissues obtained at surgery were analyzed by Congo red and immunohistochemical staining. If staining for transthyretin was positive, the entire transthyretin gene was analyzed by direct DNA sequencing. We also analyzed tenosynovial tissues from 32 autopsy cases as controls. Thirty-four patients (34.0%) with idiopathic carpal tunnel syndrome showed amyloid deposition in the tenosynovial tissue, and all amyloid showed specific immunolabeling with antitransthyretin antibody. Direct DNA sequencing of the entire transthyretin gene did not reveal any mutations, indicating that all amyloid deposits were derived form wild-type transthyretin. Statistical analysis using logistic regression showed that the prevalence of transthyretin deposition in the idiopathic carpal tunnel syndrome group was significantly higher than that in controls (odds ratio, 15.8; 95% confidence interval, 3.3-5.7), and age and male sex were independent risk factors for transthyretin amyloid deposition. Our results demonstrate that wild-type transthyretin deposition is a common cause of carpal tunnel syndrome in elderly men. It is likely that many patients develop carpal tunnel syndrome as an initial symptom of senile systemic amyloidosis.     

Investigation of the Presence of Apotipoprotein E,G lycosaminoglycans,Basement Membrane Proteins,and Protease inhibitors in Senile interstitial Amyloid of the Pituitary

The aim of our study was to test whether local- or organ-limited interstitial amyloid of the pituitary is associated with the presence of glycosaminoglycans, basement membrane proteins, protease inhibitors, and apolipoprotein E (apo E), as previously observed in other amyloid syndromes. Serial sections from amyloidotic and nonamyloidotic autopsy pituitaries of patients age 85 yr and over were stained with Congo red, Alcian blue, and, applying immunohistochemistry, with antibodies directed against fibronectin, collagen IV, laminin, apo E, α₁-antitrypsin and α₁-antichymotrypsin. Interstitial amyloid was deposited in the immediate vicinity of capillaries and around the acini of the anterior lobe. Glycosaminoglycans were found in capillaries and around the acini of both nonamyloidotic and amyloidotic glands and they were also related spatially to amyloid deposits. Immunostatining of nonamyloidotic and amyloidotic glands demonstrated the presence of fibronectin, collagen IV, and laminin, which was related to basement membranes (fibronectin, collagen IV, and laminin), interstitium, and serum (fibronectin only). In amyloidotic glands, each basement membrane protein presented with an additional spatial relationship to amyloid deposits. Apo E was found in amyloidotic cases only within the amyloid deposits. The results are consistent with the presence of glycosaminoglycans, basement membrane proteins, and apo E in local interstitial amyloid deposits of the pituitary, as previously described in other amyloid syndromes, such as inflammatory related AA-amyloidosis or Aβ-amyloidosis related to Alzheimer’s disease.     

Highly sensitive diagnosis of amyloid and various amyloid syndromes using Congo red fluorescence

In order to find how best to diagnose amyloid deposits as early as possible, the sensitivity of three different methods that can be applied to the diagnosis of amyloid in tissue sections have been compared: the Congo red staining method (CR), the combination of CR and immunocytochemistry (CRIC) and Congo red fluorescence (CRF). Tissue blocks were available from 25 patients, including 11 with immunohistochemically distinct and 3 with chemically undefined amyloid diseases. The results revealed (a) that CRF is more sensitive than either CR or CRIC, as shown qualitatively and quantitatively, (b) that CRF can therefore be utilized to track down even minute amyloid deposits, which can be missed by the other two methods; (c) that the specificity of CRF and CRIC is secured on double-stained sections by the demonstration of green birefringence (GB) of the CRF-marked and IC-marked areas; (d) that CRF can be performed on the spot by just changing the light source; and (e) that CRF is not hampered by the congruent IC chromogen overlay, which ensures the specific classification of the amyloid deposits as applied to different amyloid classes. In conclusion, CRF was demonstrated to be the most sensitive method for direct diagnosis of amyloid in tissue sections. This method can, therefore, allow the earliest diagnosis and classification of amyloid, which is a good basis for an amyloid class-specific therapy while organ damage is still minimal. Received: 29 September 1999 / Accepted: 2 December 1999     

Familial amyloidotic polyneuropathy type 1 in Kumamoto, Japan: a clinicopathologic, histochemical, immunohistochemical, and ultrastructural study

Seventeen autopsy and five biopsy cases of familial amyloidotic polyneuropathy were examined clinicopathologically, histochemically, immunohistochemically, and ultrastructurally. In the autopsy cases, amyloid deposits were predominant in the peripheral nerve tissues, autonomic nervous system, choroid plexus, cardiovascular system, and kidneys. Amyloid involvements in the anterior and posterior roots of the spinal cord, spinal ganglia, thyroid, and gastrointestinal tract were also frequent. In the cardiac conduction system, amyloid deposition was prominent in the sinoatrial node and in limbs of the intraventricular bundle. In the sural nerve biopsy, besides amyloid deposits, degenerative changes of nerve fibers and Schwann cells were detected ultrastructurally, and the morphometric analysis showed a marked reduction in the number of myelinated fibers which correlated with the clinical stage. Amyloid deposits were resistant to pretreatment with potassium permanganate in Congo red staining, and transthyretin was confirmed immunohistochemically as a major component of amyloid deposits, along with the presence of serum amyloid P-component. Besides the amyloid deposits, transthyretin was proven in the liver cells, epithelial cells of the choroid plexus, and pancreatic islet A cells, suggesting that the transthyretin produced by these cells is secreted, transferred into tissues, and deposited in situ as the major component of amyloid in this disorder.     

Amyloid in surgical pathology

Amyloid is defined as a proteinaceous tissue deposit that shows a typical green birefringence in polarized light after staining with Congo red, the presence of non-branching linear fibrils of indefinite length with a mean diameter of 10 nm, and a distinct X-ray diffraction pattern consistent with Pauling's model of a cross -fibril. Amyloid may deposit locally or may present as a systemic disease. The origin of amyloid is diverse: 25 different fibril proteins have been described so far. The precursor proteins differ from each other in their primary structures and functions. The only common denominator is the propensity to form anti-parallel cross -fibrils under certain circumstances. Early diagnosis of amyloid is still a major challenge in surgical pathology. Histological proof can be obtained using Congo-red staining and polarization microscopy. However, small deposits may be difficult to discern, and sensitivity can be improved using fluorescence microscopy. Classification of amyloid is mandatory, since amyloid is treatable and different treatment regimens are applied to different amyloid diseases. This review focuses on the epidemiology, clinical features, pathology and diagnosis of amyloid in surgical pathology.     

Wild-type transthyretin-derived amyloidosis in various ligaments and tendons

Transthyretin-derived amyloid deposition is commonly found in intercarpal ligaments of patients with senile systemic amyloidosis. However, the frequency of transthyretin-derived amyloid deposits in ligaments of other tissues remains to be elucidated. This study aimed to determine the frequency of amyloid deposition and the precursor proteins of amyloid found in orthopedic disorders. We studied 111 specimens from patients with carpal tunnel syndrome (flexor tenosynovium specimens), rotator cuff tears (rotator cuff tendon specimens), and lumbar canal stenosis (yellow ligament specimens). To identify amyloid precursor proteins, we used immunohistochemical staining with antibodies that react with transthyretin, immunoglobulin light chain, amyloid A protein, and β(2)-microglobulin. By means of Congo red staining, we identified 47 (42.3%) amyloid-positive samples, 39 of which contained transthyretin-derived amyloid (18 flexor tenosynovium specimens, 5 rotator cuff tendon specimens, and 16 yellow ligament specimens). Genetic testing and/or clinical findings suggested that all patients with transthyretin amyloid deposits did not have familial amyloidotic polyneuropathy. The occurrence of amyloid deposition in those tissues depended on age. These results suggest that transthyretin-derived amyloid deposits may occur more frequently in various ligaments and tendons than originally expected. In the future, such amyloid deposits may aid determination of the pathogenesis of ligament and tendon disorders in older patients.     

Histotopographic evidence that amyloid deposits in sclerocalcific heart valves and other chronic lesions of the cardiovascular system are related to old thrombotic material

Summary Deposition of amyloid in human sclero-calcific heart valves has been reported recently as a localized age-independant and dystrophic form of amyloidosis. Histochemical studies have shown that the deposits are permanganate resistant, contain tryptophan and P component and are immunologically unrelated to any known type of amyloid fibril protein. In this study histological observations from a series of four selected sclerotic heart valves show amyloid deposition in old thrombotic material covering fusing commissures or appositional collagen on the body of the leaflets. Similar cases from extravalvular sites have been added to the series: a partly hyalinized thrombus of the left atrium, a thrombotic aneurysm of the left ventricle, 2 thrombotic atherosclerotic aneurysms of the aorta and popliteal artery respectively, and an encapsulated haematoma of the scalp. The deposits are Congo red positive with typical green dichroism in polarized light, permanganate resistant and contain tryptophan. Electron microscopy of 3 cases displays small fibrils which are typical of amyloid.No patient showed evidence of systemic amyloidosis. The natural history of sclero-calcific valvulopathies and present observations favour the following pathogenesis: first, recurrent thrombotic deposition on thickened and fibrotic endocardium; second, degradation of a coagulation-related protein with potential during the aging of the clot with transformation into amyloid fibrils; finally, inclusion of the amyloid in sclerotic replacement tissue.     

Amyloid Deposits in Pituitaries and Pituitary Adenomas: Immunohistochemistry and In Situ Hybridization

The patterns of deposition and immunoreactivity of interstitial amyloid were studied in 11 pituitary glands obtained at autopsy and 9 surgically resected pituitary adenomas using Congo red staining and a panel of antisera directed against 5 major amyloid fibril proteins and all pituitary hormones. The deposition pattern of amyloid in pituitary glands differed from that in adenomas but all amyloid deposits showed an immunostaining with anti-amyloid λ-light chain. The remaining antisera were immunonegative.In situ hybridization using an oligodeoxyribonucleotide-probe complementary to the mRNA coding for the constant region of human λ-light chain yielded no hybridization signals in the pituitaries or pituitary adenomas, excluding local synthesis and secretion of immunoglobulins. Since no case studied suffered from generalized Aλ-amyloidosis and adsorption of immunoglobulins to the unknown amyloid fribril protein of the pituitary seems to be unlikely, crossreaction of the polyclonal antisera with an undefined antigen is probable. The similar immunostaining properties of amyloid deposits in “normal” pituitaries and pituitary adenomas suggest they both originate from the same precursor protein.     

Ultrastructural immunohistochemical localization of polyclonal IgG, C3, and amyloid P component on the congo red-negative amyloid-like fibrils of fibrillary glomerulopathy.

Renal biopsies from seven patients with Congo red-negative amyloid-like fibrillary glomerulopathy (FGP) were examined by protein A gold immuno-electron microscopy. Ultrastructurally, the fibrils in all cases exhibited positive immunostaining for IgG, both Ig light chains, C3, and amyloid P component (AP), but did not show positive immunostaining for glomerular basement membrane (GBM)-associated proteins (collagen type IV and heparan-sulfate proteoglycans) or microfibril-associated proteins (fibronectin and fibrillin). In a triple-label study, AP and IgG were colocalized along the same fibril, whereas the gold probes for the detection of collagen type IV were absent. The results suggest that the fibrils are comprised of polyclonal IgG and C3 that bind AP. AP was immunolocalized sparsely but regularly along the lamina rara interna of normal GBM. AP was absent in the fibrils in a case of diabetic glomerulopathy, was scattered randomly without specificity for the electron-dense deposits in the GBM of membranous glomerulopathy, and lined up regularly along the fibrils in amyloid deposits. FGP is an entity in which the fibrils bind AP but lack the beta-pleated sheet structure necessary for Congo red staining that is typical of amyloid.     

Evidence of the presence of amyloid substance in the blood of familial amyloidotic polyneuropathy patients with ATTR Val30Met mutation

Transthyretin (TTR) is a major amyloid fibril protein found in patients with familial amyloidotic polynuropathy (FAP) and senile systemic amyloidosis (SSA). Mainly synthesized in the live, TTR is transferred in the form of tetramer bound with thyroxine, retinol-binding protein (RBP) and lipoprotein in the blood. The aim of this study was to demonstrate the presence of amyloid substances in the blood by investigated the hemocoelom amyloid in different tissue sections from autopsies such as brain, kidney, heart and aorta arch tissue. Congo red staining was employed following by application of polarized light examination, to verify the presence of amyloid deposition in the tissues. Immunohistochemical staining was then performed to identify the specific type of amyloid deposition. Matrix-assisted laser desorption-ionization/time of flight mass spectrometry (MALDI-TOF/MS) was also used to analyze TTR mutation in FAP patients. All subjects were FAP ATTR Val30Met patients. In FAP patients, TTR amyloid deposition was found mainly in the tunica intima of the aortic arch. Interestingly, amyloid substance was found in the blood of FAP patient. Our results suggest that amyloid substance was present in the blood of FAP ATTR Val30Met patients.     

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.     

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.     

Amyloid protein of vessels in leptomeninges, cortices, choroid plexuses, and pituitary glands from patients with systemic amyloidosis

The cerebrum, cerebellum, and choroid plexuses from 16 patients with systemic amyloidosis, and the pituitary glands from 14 of these patients, were investigated histologically and immunohistochemically. Cerebrovascular amyloid (CVA) was found in the leptomeninges and cortices of six patients with systemic amyloidosis, including two patients with amyloid A protein (AA) amyloidosis related to serum amyloid A protein, one with AL amyloidosis related to immunoglobulin light chain (AL), two with familial type I amyloidotic polyneuropathy (FAP), and one with senile systemic amyloidosis (SSA). CVA protein from two patients with FAP reacted with anti-human prealbumin antibody similar to that of the visceral organs of these two patients. CVA in SSA reacted with anti-human prealbumin antibody and anti-beta protein antibody. Vascular amyloid was frequently noted in the pituitary glands and choroid plexuses of patients with systemic amyloidosis, and was found to be identical to that in the visceral organs (heart, kidney, and intestine) of these patients. CVA in the leptomeninges and cortices from two patients with AA amyloidosis and one with AL amyloidosis reacted with anti-beta protein monoclonal antibody but not with anti-human AA monoclonal antibody, anti-human A lambda antisera, and anti-human A kappa antisera. We suggest that amyloid proteins of AA and AL amyloidosis do not readily accumulate in the vessels in the leptomeninges and cortices even though the proteins circulate, and that beta protein is not derived from a serum precursor.