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.     

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.     

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).     

A novel tool for detecting amyloid deposits in systemic amyloidosis in vitro and in vivo

We synthesized (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) and used this compound to detect amyloid fibrils in autopsy and biopsy samples from patients with localized amyloidosis, such as familial prion disease, and systemic amyloidosis, such as familial amyloidotic polyneuropathy, amyloid A (AA) amyloidosis, light chain (AL) amyloidosis, and dialysis-related amyloidosis. BSB showed reactions in all Congo red-positive and immunoreactive regions of the samples examined in the study, and some amyloid fibrils in the tissues could be detected more precisely with BSB than with the other methods. In the mouse model of AA amyloidosis, injected BSB reacted with amyloid in all regions in the serial sections in which Congo red staining was positive. A highly sensitive 27-MHz quartz crystal microbalance analysis revealed that BSB showed a significant affinity for amyloid fibrils purified from familial amyloidotic polyneuropathy and dialysis-related amyloidosis samples and suppressed formation of transthyretin amyloid in vitro. These results suggest that BSB may become a valuable tool for detection of amyloid deposits in amyloidosis and of the mechanism of amyloid formation.     

Die Amyloidosen des Berliner Medizinhistorischen Museums der Charité

The Berliner Medizinhistorische Museum (Berlin Museum of Medical History) of the Charité is located in its own separate building and was officially opened in 1899. It currently houses 10,000 specimens, of which 23 were labelled with the diagnosis “amyloid” or “amyloidosis”. In this retrospective study we aimed to histologically verify the diagnosis, classify the amyloid deposits immunohistochemically and correlate the type of amyloid with clinico-pathological data. The specimens were obtained between 1866 and 1987 and included 17 kidneys, five spleens and one liver. The diagnosis could be confirmed histologically using Congo red staining and polarization microscopy in 22 specimens. However, the diagnosis could not be confirmed in the oldest specimen, which had been labelled by Rudolf Virchow himself. Immunohistochemically amyloid was classified as either AA amyloidosis (19 cases) or AL amyloidosis (two cases). Tuberculosis was the most common cause of AA amyloidosis. This study shows that a surgical pathological re-evaluation of historical specimens can verify the original diagnosis. This is historically fascinating and also offers a valuable addition to student teaching.     

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.     

Quantitative analysis of interstitial mast cells in AA and AL renal amyloidosis

Eighteen renal biopsy specimens obtained from patients with AA-type renal amyloidosis (AA) and 11 from patients with AL-type renal amyloidosis (AL), for whom both light and electron microscopy as well as immunofluorescence microscopy and full clinical data were available, were examined quantitatively. The cases were selected on the basis of immunohistochemical studies. As a control, we used 10 biopsy specimens from the kidneys removed because of trauma. Morphometric investigations were carried out by a computer image analysis system to find an answer to the question of whether mast cells can correlate with tubulointerstitial fibrosis in AA and AL renal amyloidosis, and to examine the relationship between mast cells and interstitial alpha-smooth muscle actin (alpha-SMA) expression and interstitial infiltrates. The morphometric study revealed that the mean values of the interstitial tryptase-positive cells, expression of alpha-SMA, interstitial volume, CD68+, CD45RB+, CD43+ and CD20+ cells were increased in AA as compared with the AL group, most of them significantly. Most of these parameters were also significantly increased in both AA and AL patients as compared with the control group. In both the AA group and the AL group, there existed some significant positive correlations between interstitial tryptase-positive cells and interstitial expression of alpha-SMA, interstitial volume and CD68+ cells. Interestingly, in AA cases, but not in AL cases, we noted a significant relationship between interstitial tryptase-positive cells and CD43+ cells. Our findings demonstrate that mast cells belong to the constitutive cell types in the interstitium in renal amyloidosis, in particular in amyloid type A. In addition, in both the AA group and the AL group, the significant positive correlations between interstitial mast cell count and relative interstitial volume and interstitial expression of alpha-SMA suggest that these cells play a role in the development of interstitial fibrosis.     

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.     

AA-type amyloidosis associated with non-Hodgkin's lymphoma: a case report

Amyloid-associated protein (AA)-type systemic amyloidosis has been referred to as secondary amyloidosis because it is secondary to an associated inflammatory condition. It is extremely rare in patients with non-Hodgkin's lymphoma (NHL). Here we report an autopsy case of follicular small cleaved cell lymphoma with focal large B-cell lymphoma transformation in association with systemic AA-type amyloidosis. Formalin-fixed, paraffin-embedded tissues from autopsy and the patient's previous surgical specimen were studied by Congo red stain; electron microscopy; and immunostaining with antibodies against AA protein, P component, and kappa and lambda light chains. There was a marked AA amyloid deposition in the glomeruli of both kidneys, the retroperitoneal lymphoma mass, the blood vessels, the adrenal glands, and the adipose tissues. The patient's previous surgical specimens were negative for amyloid. We propose that this patient's systemic AA-type amyloidosis developed along the course of his NHL.     

Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy

Amyloid fat pad aspiration specimens for cases with a clinical suspicion of amyloid typically are stained with Congo red and examined by brightfield microscopy. Congophilia with apple-green birefringence by polarization microscopy (PM) is considered diagnostic for amyloid. Examination of Congo red-stained slides by fluorescent microscopy (FM) is considered by some to be a more sensitive detection method. In this study, we assessed the utility of this technique in cytopathology archival slides from abdominal fat pad aspirations previously stained with Congo red dye. Seventy-eight cases of abdominal fat pad aspirations collected during the last 5 yr and stained with the Congo red procedure were obtained from archival files. Additionally, 20 adipose tissue material slides prepared from the surgical pathology specimens were examined as controls. One representative smear was examined in each case using FM equipped with rhodamine excitation/absorption (540/570 nm) filters. Relevant clinical information was obtained in all cases. Twelve cases (15.4%) of the 78 fat pad aspiration cases were reported originally as positive by Congo red stain using polarization and apple-green birefringence as diagnostic criteria. On review, four cases were deemed unsatisfactory. By FM examination 29 of the 74 (39.2%) cases were reclassified as positive for amyloid. The results were confirmed by immunohistochemical stain for amyloid P protein and electron microscopy. A number of similar distinct fluorescence and immunohistochemical patterns were recognized in the positive cases. Minimally weak fluorescence in the adipose tissue was observed in the control cases. The use of FM in Congo red-stained fat pad smears can improve the detection of amyloid in cytology preparations.     

Subepithelial argyrophilic spicular structures in renal amyloidosis—An aid in diagnosis: Pathogenic considerations

Eighteen cases of amyloidosis with renal involvement were classified utilizing clinical and laboratory data as to the most likely major amyloid fibril protein type and studied as to their histological, tinctorial, immunofluorescence, and electron microscopic features. No differences could be appreciated between the AA and AL types of amyloidosis. Immunofluorescence did not aid in the diagnosis of amyloidosis and was confusing in some cases owing to apparent absorption of serum proteins. Subepithelial spicular structures were noted in the glomerular capillary loops in 14 of 18 cases (78 per cent), and similar structures were found related to tubular epithelial cells in six of these cases and were related to the parietal epithelium of Bowman's membrane in one case. These spicular structures were a valuable aid in the diagnosis of early amyloidosis by light microscopy, but electron microscopy was essential for confirmation. We postulate that because of the intense staining quality of spicular structures using argyrophilic techniques, these spicules result from a unique interaction between amyloid fibrils and locally produced substances, most probably renal epithelial basement membrane glycoprotein.     

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.     

肾脏早期淀粉样变临床病理特点

目的 探讨光学显微镜尚未表现明显特点的早期肾淀粉样变(amyloidosis,AL)的临床病理特点及诊断方法。方法 回顾性分析该院1994-2001年间诊断的肾AL病例,重点对其中15例光镜未能及时诊断而通过透射电镜诊断的早期AL病例,进行临床病理特点的分析,并对其肾活检组织进行了轻链蛋白(κ、λ)的免疫电镜定位检测。结果 15例均为早期肾AL,发病年龄以中老年为主,突出表现为肾病综合征,偶见镜下血尿及高血压,肾功能正常。多数以肾脏病为初诊症状。肾活检组织光镜观察病变表现轻微,可有系膜轻度增生或基底膜空泡变性及轻度增厚;免疫荧光表现不一,部分病例全部阴性,部分表现免疫球蛋白及补体沿系膜区或毛细血管壁不同程度的沉积,均有单一品种的轻链蛋白沉积;光镜初步诊断为肾小球微小病变4例,轻度系膜增生性肾小球肾炎5例,I期膜性肾病5例和1例管型肾病;电镜观察可见系膜区、基底膜及小动脉壁上的淀粉样纤维分布;补作刚果红染色时,呈阳性反应;免疫电镜可观察到轻链蛋白被标记于淀粉样纤维部位,证实15例均为轻链型AL。结论 电镜检查是发现及早期诊断肾淀粉样变的重要手段,单一品种的轻链蛋白沉积有一定的诊断意义,结合免疫电镜检查可进一步确诊和分型。     

Secondary (AA) amyloidosis in cystic fibrosis. A report of three cases

The authors report the pathologic features of three cases of amyloidosis associated with cystic fibrosis. Renal biopsy led to the diagnosis (case 1) or suspicion (case 2) of amyloidosis in patients who were 23 and 21 years old, respectively. The third patient died at age 22 years, and amyloidosis was not discovered until autopsy. Immunohistochemical staining and potassium-permanganate pretreatment of histologic sections in all three cases provided evidence that the amyloid seen in these patients is of the secondary (AA) type. Congo red staining in each case and electron microscopy in case 1 confirmed the initial diagnosis of amyloidosis. A markedly elevated serum amyloid A protein (160 micrograms/mL; normal less than 1 microgram/mL) in case 1 indicated the presence of large quantities of the precursor protein from which the AA fibrils of secondary amyloid are derived. The kidneys, spleen, and liver contained amyloid deposits in autopsy material from all three cases. Involvement of other organs by amyloid was variable. Review of autopsy material in Boston from 23 additional cystic fibrosis patients with long-term survival did not reveal any evidence of amyloidosis. It appears that secondary amyloidosis is emerging as a significant, although rare, complication of cystic fibrosis as greater numbers of these patients survive into adulthood.     

Sensitivity and specificity of Congo red staining according to Romhányi. Comparison with Puchtler's or Bennhold's methods

The sensitivity and specificity of various Congo red staining methods is very important in the diagnosis of amyloidosis. When using a less sensitive staining method, some true positive cases of amyloidosis remain undetected. A more highly specific method potentially detects more cases and reveals amyloidosis in an earlier stage of deposition. In this paper, the Congo red staining method according to Romhányi is discussed in comparison with Puchtler's and Bennhold's methods. Using Romhányi's technique, there is no alcoholic differentiation, and thus no dye molecules are washed off the amyloid filaments. The binding of the oriented dye molecules is optimal for polarization microscopy. With this method, the polar hydrophilic mounting medium, gum Arabic is used. Mounted in this carbohydrate-containing, hydrophilic medium, the Congo red molecules are oriented parallel to the surface of the amyloid filaments and the sign is linear positive, corresponding to an additive character of topo-optical staining reactions. Otherwise, the Congo red molecules are oriented perpendicular to the surface of collagen, reducing the intensity of birefringence and even inducing an inversion of the original sign of the collagen birefringence. With alcoholic differentiation, Congo red dye molecules are extracted and this decreases the birefringence of amyloid deposits, i.e. minimal amyloid deposits may be missed. Using the apolar hydrophobic mounting medium, Canada balsam, an axis-parallel arrangement of Congo red dye molecules on the surface of collagen fibers and amyloid will occur, resulting in an additive topo-optical reaction with a green polarization color and a false positive diagnosis of amyloidosis ("phantom amyloidosis").     

Sensitivity and specificity of Congo red staining according to Romhányi. Comparison with Puchtler's or Bennhold's methods

The sensitivity and specificity of various Congo red staining methods is very important in the diagnosis of amyloidosis. When using a less sensitive staining method, some true positive cases of amyloidosis remain undetected. A more highly specific method potentially detects more cases and reveals amyloidosis in an earlier stage of deposition. In this paper, the Congo red staining method according to Romhányi is discussed in comparison with Puchtler's and Bennhold's methods. Using Romhányi's technique, there is no alcoholic differentiation, and thus no dye molecules are washed off the amyloid filaments. The binding of the oriented dye molecules is optimal for polarization microscopy. With this method, the polar hydrophilic mounting medium, gum Arabic is used. Mounted in this carbohydrate-containing, hydrophilic medium, the Congo red molecules are oriented parallel to the surface of the amyloid filaments and the sign is linear positive, corresponding to an additive character of topo-optical staining reactions. Otherwise, the Congo red molecules are oriented perpendicular to the surface of collagen, reducing the intensity of birefringence and even inducing an inversion of the original sign of the collagen birefringence. With alcoholic differentiation, Congo red dye molecules are extracted and this decreases the birefringence of amyloid deposits, i.e. minimal amyloid deposits may be missed. Using the apolar hydrophobic mounting medium, Canada balsam, an axis-parallel arrangement of Congo red dye molecules on the surface of collagen fibers and amyloid will occur, resulting in an additive topo-optical reaction with a green polarization color and a false positive diagnosis of amyloidosis (“phantom amyloidosis”).     

The pattern of amyloidosis in Malaysia

Congo red screening of routine biopsies at the University Hospital Kuala Lumpur revealed the following categories of amyloidosis: systemic AL (5.9%); systemic AA (3.2%); isolated atrial (14%); primary localized cutaneous (7.5%); other primary localized deposits (3.2%); localized intratumour (58%); and dystrophic (8.6%). Unlike in the West, AA amyloidosis in this population was usually secondary to leprosy or tuberculosis. Liver involvement in AL amyloidosis was shown to exhibit a sinusoidal pattern and differed from the vascular pattern of AA amyloidosis. Within the category of AA amyloidosis, there were two patterns of renal involvement--glomerular and vascular, with the glomerular pattern carrying a more ominous clinical picture. Notable among the localized amyloidoses were isolated atrial amyloidosis complicating chronic rheumatic heart disease, intratumour amyloidosis within nasopharyngeal carcinomas and dystrophic amyloidosis which occurred in fibrotic tissues.     

激光显微切割联合质谱分析继发淀粉样变性患者肾组织SAA蛋白

目的利用激光显微切割联合质谱(LMD/MS)技术分析强直性脊柱炎(AS)合并继发性淀粉样变患者肾活检组织标本血清淀粉样物质A(SAA)蛋白亚型及氨基酸突变序列。方法甲醛固定肾活检组织,脱蜡后行刚果红染色,选取刚果红染色阳性区域进行质谱分析,通过数据分析软件对质谱结果进行整合评估,并将患者SAA蛋白氨基酸序列与变异蛋白数据库氨基酸序列进行比对确定是否有变异蛋白。结果质谱鉴定到高丰度的SAA1及SAA2蛋白,同时有血清淀粉样蛋白P及载脂蛋白E,数据库比对未检测到SAA1及SAA2蛋白的变异序列。结论本研究首次鉴定到了AS合并淀粉样变肾组织中的SAA1及SAA2蛋白,丰富了AS淀粉样变的发病机制,为将来AA型淀粉样变性的精准分型提供新的方法。     

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.