Structural Typing of Systemic Amyloidoses by Luminescent-Conjugated Polymer Spectroscopy
2009; Elsevier BV; Volume: 176; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2010.080797
ISSN1525-2191
AutoresK. Peter R. Nilsson, Kristian Ikenberg, Andreas Åslund, Sophia Fransson, Peter Konradsson, Christoph Röcken, Holger Moch, Adriano Aguzzi,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoMost systemic amyloidoses are progressive and lethal, and their therapy depends on the identification of the offending proteins. Here we report that luminescent-conjugated thiophene polymers (LCP) sensitively detect amyloid deposits. The heterodisperse polythiophene acetic acid derivatives, polythiophene acetic acid (PTAA) and trimeric PTAA, emitted yellow-red fluorescence on binding to amyloid deposits, whereas chemically homogeneous pentameric formic thiophene acetic acid emitted green-yellow fluorescence. The geometry of LCPs modulates the spectral composition of the emitted light, thereby reporting ligand-induced steric changes. Accordingly, a screen of PTAA-stained amyloid deposits in histological tissue arrays revealed striking spectral differences between specimens. Blinded cluster assignments of spectral profiles of tissue samples from 108 tissue samples derived from 96 patients identified three nonoverlapping classes, which were found to match AA, AL, and ATTR immunotyping. We conclude that LCP spectroscopy is a sensitive and powerful tool for identifying and characterizing amyloid deposits. Most systemic amyloidoses are progressive and lethal, and their therapy depends on the identification of the offending proteins. Here we report that luminescent-conjugated thiophene polymers (LCP) sensitively detect amyloid deposits. The heterodisperse polythiophene acetic acid derivatives, polythiophene acetic acid (PTAA) and trimeric PTAA, emitted yellow-red fluorescence on binding to amyloid deposits, whereas chemically homogeneous pentameric formic thiophene acetic acid emitted green-yellow fluorescence. The geometry of LCPs modulates the spectral composition of the emitted light, thereby reporting ligand-induced steric changes. Accordingly, a screen of PTAA-stained amyloid deposits in histological tissue arrays revealed striking spectral differences between specimens. Blinded cluster assignments of spectral profiles of tissue samples from 108 tissue samples derived from 96 patients identified three nonoverlapping classes, which were found to match AA, AL, and ATTR immunotyping. We conclude that LCP spectroscopy is a sensitive and powerful tool for identifying and characterizing amyloid deposits. Amyloidoses are diseases of disparate etiologies characterized by extracellular proteinaceous deposits in tissues and organs. These deposits, termed "amyloids," result from misfolding and/or partial unfolding of proteins followed by their ordered aggregation. At least 27 different proteins have been reported to form disease-associated amyloids in vivo1Enqvist S Sletten K Stevens FJ Hellman U Westermark P Germ line origin and somatic mutations determine the target tissues in systemic AL-amyloidosis.PLoS ONE. 2007; 2: e981Crossref PubMed Scopus (32) Google Scholar, and there is evidence that any given polypeptide can be induced to form amyloid in vitro under appropriate conditions.2Chiti F Dobson CM Protein misfolding, functional amyloid, and human disease.Annu Rev Biochem. 2006; 75: 333-366Crossref PubMed Scopus (5112) Google Scholar It has been suggested that amyloid may represent a primordial state for all proteins, which would be always attained unless specific antiamyloidogenic factors (including chaperones and disaggregases) prevent or revert its formation. Accordingly, formation of amyloids may be much more prevalent than currently appreciated3Maji SK Perrin MH Sawaya MR Jessberger S Vadodaria K Rissman RA Singru PS Nilsson KPR Simon R Schubert D Eisenberg D Rivier J Sawchenko P Vale W Riek R Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.Science. 2009; 325: 328-332Crossref PubMed Scopus (767) Google Scholar and may contribute to several diseases whose etiology is hitherto unclear.4Aguzzi A Cell biology: Beyond the prion principle.Nature. 2009; 459: 924-925Crossref PubMed Scopus (161) Google Scholar Some amyloids are highly selective in their organ tropism and lead to localized amyloidosis. Two highly prevalent organ-specific amyloidoses are caused by the Aβ peptide5Aguzzi A Haass C Games played by rogue proteins in prion disorders and Alzheimer's disease.Science. 2003; 302: 814-818Crossref PubMed Scopus (200) Google Scholar and islet amyloid polypeptide, which are associated with Alzheimer's disease and type 2 diabetes, respectively. Prion diseases, which go along with aggregation of PrPSc, can be regarded as transmissible amyloidoses of the nervous and lymphoreticular system.6Aguzzi A Polymenidou M Mammalian prion biology: one century of evolving concepts.Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar Other amyloidogenic proteins deposit in many organs. Such "systemic" amyloidoses can be neoplastic, inflammatory, or genetic in origin and present as chronic progressive diseases leading to lethal heart and renal failure if untreated. Depending on the biochemical nature of the amyloid in question, the average survival can be as short as 12 months, or can span 7–15 years. Therapeutic options for amyloidoses include aggressive treatments directed at reducing precursor protein production and/or at inhibiting the extracellular deposition of amyloid fibrils, eg, by pharmacological immunosuppression or even by surgical replacement of the organ synthesizing the precursor protein. The indication for each of these therapies, and their prognostic prospect, depend on the specific type of amyloid.7Dember LM Emerging treatment approaches for systemic amyloidoses.Kidney Int. 2005; 68: 1377-1390Crossref PubMed Scopus (34) Google Scholar, 8Comenzo RL Current and emerging views and treatments of systemic immunoglobulin light-chain (AL) amyloidosis.Contrib Nephrol. 2007; 153: 195-210Crossref PubMed Scopus (27) Google Scholar As optimal outcomes rely on early therapy, sensitive methods for diagnosis and selective technologies for identification of amyloid subtypes are of greatest importance. Systemic amyloidoses are classified according to the chemical nature of the predominant amyloid constituent, with the most common amyloids being AL, AA, and ATTR.9Westermark P Benson MD Buxbaum JN Cohen AS Frangione B Ikeda S Masters CL Merlini G Saraiva MJ Sipe JD A primer of amyloid nomenclature.Amyloid. 2007; 14: 179-183Crossref PubMed Scopus (270) Google Scholar, 10Obici L Perfetti V Palladini G Moratti R Merlini G Clinical aspects of systemic amyloid diseases.Biochim Biophys Acta. 2005; 1753: 11-22Crossref PubMed Scopus (213) Google Scholar AL amyloid consists of Ig light chains, or fragments thereof, and is associated with various B cell lymphoproliferative disorders including multiple myeloma.10Obici L Perfetti V Palladini G Moratti R Merlini G Clinical aspects of systemic amyloid diseases.Biochim Biophys Acta. 2005; 1753: 11-22Crossref PubMed Scopus (213) Google Scholar, 11Sanchorawala V Light-chain (AL) amyloidosis: diagnosis and treatment.Clin J Am Soc Nephrol. 2006; 6: 1331-1341Crossref Scopus (177) Google Scholar, 12Sanchorawala V Blanchard E Seldin DC O'Hara C Skinner M Wright DG AL amyloidosis associated with B-cell lymphoproliferative disorders: frequency and treatment outcomes.Am J Hematol. 2006; 81: 692-695Crossref PubMed Scopus (50) Google Scholar, 13Picken MM New insights into systemic amyloidosis: the importance of diagnosis of specific type.Curr Opin Nephrol Hypertens. 2007; 16: 196-203Crossref PubMed Scopus (96) Google Scholar AA amyloid is derived from serum amyloid A (AA) protein and is associated with chronic immune activation, as in chronic infections, autoimmune or hereditary inflammatory diseases, or cancer.14Röcken C Shakespeare A Pathology, diagnosis and pathogenesis of AA amyloidosis.Virchows Arch. 2002; 440: 111-122Crossref PubMed Scopus (185) Google Scholar, 15Lachmann HJ Hawkins PN Systemic amyloidosis.Curr Opinion Pharmacol. 2006; 6: 1-7Crossref Scopus (83) Google Scholar ATTR systemic amyloid consists of transthyretin deposits and occurs sporadically or in association with transthyretin mutations that enhance protein misfolding and fibril formation.16Pitkänen P Westermark P Cornwell GG Senile systemic amyloidosis.Am J Path. 1984; 117: 391-399PubMed Google Scholar, 17Johansson B Westermark P Senile systemic amyloidosis: a clinico-pathological study of twelve patients with massive amyloid infiltration.Int J Cardiol. 1991; 32: 83-92Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 18Hawkins PN Hereditary systemic amyloidoses with renal involvement.J Nephrol. 2003; 16: 443-448PubMed Google Scholar, 19Ando Y Nakamura M Araki S Transthyretin-related familial amyloidotic polyneuropathy.Arch Neurol. 2005; 62: 1057-1062Crossref PubMed Scopus (333) Google Scholar Amyloid deposits consist of fibrils of 7–10 nm in diameter displaying a cross β-pleated sheet conformation. This common structural property has enabled the development of generic amyloid ligands, such as Congo Red and thioflavins. The gold-green birefringence of Congo Red-stained amyloid is commonly considered the diagnostic gold standard. However, small amyloid ligands do not differentiate between amyloid subtypes. Hence, the biochemical classification of amyloids must be pursued by other means. In hereditary amyloidoses, certainty can be attained by identifying causative mutations in the genes encoding the respective precursor protein. Mass spectrometric identification of amyloid-derived peptides can be diagnostic, but it is far too cumbersome for routine clinical diagnostics. Therefore, immunohistochemical stains are typically used for differentiating amyloid subtypes.13Picken MM New insights into systemic amyloidosis: the importance of diagnosis of specific type.Curr Opin Nephrol Hypertens. 2007; 16: 196-203Crossref PubMed Scopus (96) Google Scholar However, immunohistochemistry is fraught with specific problems. Most antibodies penetrate only poorly the compact amyloid structures. Also, many amyloids incorporate Igs and complement-derived opsonins, which can give rise to false-positive stains with diagnostic antibodies and—in worst-case scenarios—may lead to misdiagnoses.13Picken MM New insights into systemic amyloidosis: the importance of diagnosis of specific type.Curr Opin Nephrol Hypertens. 2007; 16: 196-203Crossref PubMed Scopus (96) Google Scholar, 20Lachmann HJ Booth DR Booth SE Bybee A Gilbertson JA Gillmore JD Pepys MB Hawkins PN Misdiagnosis of hereditary amyloidosis as AL (primary) amyloidosis.N Engl J Med. 2002; 346: 1786-1791Crossref PubMed Scopus (535) Google Scholar, 21Novak L Cook WJ Herrera GA Sanders PW AL-amyloidosis is underdiagnosed in renal biopsies.Nephrol Dial Transplant. 2004; 19: 3050-3053Crossref PubMed Scopus (67) Google Scholar, 22Kebbel A Röcken C Immunohistochemical classification of amyloid in surgical pathology revisited.Am J Surg Pathol. 2006; 30: 673-683Crossref PubMed Scopus (120) Google Scholar, 23Satoskar AA Burdge K Cowden DJ Nadasdy GM Hebert LA Nadasdy T Typing of amyloidosis in renal biopsies—diagnostic pitfalls.Arch Pathol Lab Med. 2007; 131: 917-922PubMed Google Scholar We have previously reported that luminescent-conjugated polymers (LCPs) interact with amyloid fibrils and amyloid deposits in tissue sections.24Nilsson KPR Herland A Hammarström P Inganäs O Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation.Biochemistry. 2005; 44: 3718-3724Crossref PubMed Scopus (159) Google Scholar, 25Herland A Nilsson KPR Olsson JDM Hammarström P Konradsson P Inganäs O Synthesis of a regioregular zwitterionic conjugated oligoelectrolyte, usable as an optical probe for detection of amyloid fibril formation at acidic pH.J Am Chem Soc. 2005; 127: 2317-2323Crossref PubMed Scopus (132) Google Scholar, 26Nilsson KPR Hammarström P Ahlgren F Herland A Schnell EA Lindgren M Westermark GT Inganäs O Conjugated polyelectrolytes-conformation-sensitive optical probes for staining and characterization of amyloid deposits.Chembiochem. 2006; 7: 1096-1104Crossref PubMed Scopus (115) Google Scholar Whereas conventional amyloid ligands, such as Congo Red and thioflavin derivatives, are sterically rigid and fluoresce with defined spectra, LCPs are composed of rotationally flexible polythiophene chains that fluoresce in different colors depending on their geometry. Therefore, the emission spectra recorded from LCPs reflect the conformation of their backbones. The interaction of LCPs with protein aggregates imposes rotational constraints leading to spectroscopic signatures indicative of specific supramolecular structures. This phenomenon allows for discriminating mouse-passaged prion strains that may solely differ in their structure.27Sigurdson CJ Nilsson KPR Hornemann S Manco G Polymenidou M Schwarz P Leclerc M Hammarström P Wuthrich K Aguzzi A Prion strain discrimination using luminescent conjugated polymers.Nat Methods. 2007; 4: 1023-1030Crossref PubMed Scopus (233) Google Scholar Analogously, we were able to differentiate multiple heterogeneous types of Aβ deposits in the brain of transgenic mice.28Nilsson KPR Åslund A Berg I Nyström S Konradsson P Herland A Inganäs O Stabo-Eeg F Lindgren M Westermark GT Lannfelt L Nilsson LN Hammarström P Imaging distinct conformational states of amyloid-β fibrils in Alzheimer's disease using novel luminescent probes.ACS Chem Biol. 2007; 2: 553-560Crossref PubMed Scopus (167) Google Scholar Here we tested the usefulness of three anionic LCPs, polythiophene acetic acid (PTAA), trimeric polythiophene acetic acid (tPTAA), and pentameric formic thiophene acetic acid (p-FTAA) for the typing of human systemic amyloidoses. We found that the analysis of spectral signatures of anionic LCPs provide complementary information to conventional techniques regarding the nature of the amyloid deposits and that LCP fluorescence provides a more precise readout than the polarized absorbance of Congo Red. Hence, LCPs represent a complementary tool for rapid and accurate diagnosis of systemic amyloidoses. Patients diagnosed with amyloidosis during the period from 1996 to 2007 were identified in the archives of the Institute of Surgical Pathology (Zurich, Switzerland). We chose 54 paraffin-embedded tissue blocks with conspicuous amyloid deposits from 42 patients, including 10 women and 32 men. Clinical information was retrieved from hospital records. The mean age at diagnosis was 70.5 years (range, 17–96 years). The tissue samples of the 42 patients with systemic amyloidosis included heart (n = 27), kidney (n = 4), tongue (n = 1), esophagus (n = 1), seminal vesicle (n = 1), stomach (n = 2), soft tissue (n = 2), lymph node (n = 1), lung (n = 2), sigma colon (n = 1), small intestine (n = 1), duodenum (n = 2), conjunctiva (n = 1), liver (n = 1), colon (n = 1), prostate (n = 1), thyroid (n = 1), adrenal gland (n = 1), parathyroid (n = 1), and joint capsule (n = 2). All tissue specimens were fixed in 4% formalin and embedded in paraffin. The present study was performed according to the ethical rules for establishments of novel diagnostic tests in the Kanton Zurich under strictly unlinked-anonymous conditions. A "validation set" of amyloid specimens was retrieved from the Amyloid Registry of the Charité University Hospital (Berlin, Germany) and included 54 patients with histologically confirmed amyloid (37 males, 17 females; mean age, 66.4 years; range, 42–86 years). The tissue samples were obtained from the heart (17 cases), colon and rectum (12), stomach (5), liver (4), kidney (3), iliac crest (3), lung (2), skin (2), lymph node (1), small intestine (1), tendons (1), thyroid gland (1) tongue (1), and ureter (1). All specimens had been fixed in formalin and embedded in paraffin. In each sample selected for this study, the presence of amyloid was confirmed by histological examination of the birefringence of deparaffinized, Congo Red-stained sections. The quality of the tissue material and amount of amyloid deposits were documented for each sample. Amyloid subtypes were classified by immunohistochemistry with a broad panel of reagents including commercially available primary monoclonal or polyclonal antibodies. Antibodies against AA (diluted 1/500; monoclonal), transthyretin (TTR) (1/800; polyclonal), human kappa light chains (κ; polyclonal), and lambda light chains (λ; polyclonal) were purchased from Dako (Glostrup, Denmark). Primary monoclonal antibodies against human kappa (κ) and lambda (λ) light chains (1/50) were purchased from BMA Biomedicals (Augst, Switzerland). Staining of the tissue microarrays (TMAs) was performed at the Institute of Pathology of the Basel University Hospital (Basel, Switzerland). Immunohistochemistry was performed on deparaffinized formalin-fixed tissue using an automated staining system (Ventana Medical Systems, Tucson, AZ) or manually. Slides were heated with cell conditioner for antigen retrieval and endogenous biotin was blocked with a standard kit. Immunoreactivity was visualized with iVIEW DAB detection kit. Slides were counterstained with hematoxylin before glass coverslipping, and interpreted according to recent recommendations.29Linke RP Oos R Wiegel NM Nathrath WB Classification of amyloidosis: misdiagnosing by way of incomplete immunohistochemistry and how to prevent it.Acta Histochem. 2006; 108: 197-208Crossref PubMed Scopus (74) Google Scholar The amyloid deposits of the "validation set" were classified immunohistochemically as described elsewhere.30Eriksson M Buttner J Todorov T Yumlu S Schonland S Hegenbart U Kristen AV Dengler T Lohse P Helmke B Schmidt H Röcken C Prevalence of germline mutations in the TTR gene in a consecutive series of surgical pathology specimens with ATTR amyloid.Am J Surg Pathol. 2009; 33: 58-65Crossref PubMed Scopus (33) Google Scholar The synthesis of PTAA (mean mol. wt., mol. wt. = 3 kDa), tPTAA (mol. wt. = 1.5 kDa) and p-FTAA (mol. wt. = 615 Da) have been reported elsewhere.31Åslund A Herland A Hammarström P Nilsson KPR Jonsson B-H Konradsson P Inganäs O Studies of luminescent conjugated polythiophene derivatives: enhanced spectral discrimination of protein conformational states.Bioconjug Chem. 2007; 18: 1860-1868Crossref PubMed Scopus (69) Google Scholar, 32Åslund A Sigurdson CJ Klingstedt T Grathwohl S Bolmont T Dickstein DL Glimsdal E Prokop S Lindgren M Konradsson P Holtzman DM Hof PR Heppner FL Gandy S Jucker M Aguzzi A Hammarström P Nilsson KPR Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses.ACS Chem Biol. 2009; 4: 673-684Crossref PubMed Scopus (239) Google Scholar, 33Ding LJM Roman LS Andersson MR Inganäs O Photovoltaic cells with a conjugated polyelectrolyte.Synt Met. 2000; 110: 133-140Crossref Scopus (78) Google Scholar Paraffin-embedded formalin fixed sections were deparaffinized. After rehydration with deionized water, the sections were equilibrated in PBS (p-FTAA staining) or 100 mmol/L sodium carbonate at pH = 10 (PTAA and tPTAA staining). LCPs were diluted in incubation buffer (10 μg/ml), added to the tissue sections, incubated for 30 minutes at room temperature, and removed by washes with incubation buffer. Images and spectra were recorded with a Zeiss Axioplan 2 microscope fitted with a Spectraview 4.0 (Applied Spectral Imaging, Migdal, Israel) and a Spectra-Cube (interferometrical optical head SD 300) module with cooled CCD-camera, through a 405/30-nm (LP 450) bandpass filter. The data were processed with SpectraView 3.0 EXPO software. Spectra were collected from LCP stained amyloid deposits (5–10 deposits for each sample and eight individual spots from each deposit) and other fluorescent entities. Fluorescent spectral unmixing (SUN) was performed using the function in the software. The spectra recorded for amyloid deposits in positive samples were used for SUN analysis of the negative control sample. An amyloid TMA was constructed to investigate the LCP selectivity for amyloid. The TMA approach allows the direct comparison between many different subtypes of amyloids and negative controls within one small tissue cylinder. Paraffin-embedded tissues were selected for the TMA construction on the basis of Congo Red-stained tissue sections. Only tissue blocks with conspicuous amyloid deposits were used. Up to four cores (0.6 mm in diameter; length: 3–4 mm) were taken from each representative tissue block to increase the possibility of sufficient amyloid deposits in the tissue cores of the TMA. Cores from four different tissues without amyloidosis (heart, pancreas, spleen, and kidney) were included as negative control. The cores were transferred to the recipient paraffin block as described previously.34Bubendorf L Nocito A Moch H Sauter G Tissue microarray (TMA) technology: miniaturized pathology archives for high-throughput in situ studies.J Pathol. 2001; 195: 72-79Crossref PubMed Scopus (325) Google Scholar Four-micrometer-thick sections were cut for further analysis. There were sufficient amyloid deposits with positivity for both Congo Red and immunohistochemistry in tissue cores of 20 patients. For spectral collection of PTAA bound to amyloid aggregates, tissue sections were analyzed as follows: eight individual spots within each of 5–10 plaques from each case were examined, yielding 30–50 measurements per case. The fluorescent intensity ratios were calculated (R538 nm/Emax and R538 nm/642 nm), and mean and SD were recorded for each spectral ratio for each individual. To make up homogeneous groups of objects (classes) on the basis of their description by the two ratios, a k-means clustering analysis was performed (XLSTAT, 2008). In addition an unpaired, two-tailed Student's t-test was performed using mean values of single cases as observations (GraphPad Prism 5). The raw data from each individual case and the results from the statistical evaluations are provided as Supplemental Tables S1–S14 at http://ajp.amjpathol.org. Amyloid deposits in 54 tissue samples from 42 patients were typed by immunohistochemistry on TMAs and on selected conventional tissue sections (Tables 1 and 2). For the nomenclature of amyloid and amyloidoses, we followed the recommendations by the International Nomenclature Committee on Amyloidosis. We stained TMA sections with AA, AL, or ATTR antibodies and scored immunohistochemical signals as interpretable only when they colocalized with unambiguous Congo Red signals.Table 1Clinical Description and Tinctorial Properties of TMA SpecimensPatientOrganPrimary diagnosisAge/SexCongo Red*Positive or negative staining are represented with + or −, respectively.IHC†Amyloid subtype according to immunohistochemistry (IHC).PTAA‡The numbers 1, 2, or 3 refer to the spectral classification of PTAA tingibility.tPTAA*Positive or negative staining are represented with + or −, respectively.p-FTAA*Positive or negative staining are represented with + or −, respectively.1HeartHeart failure80/M+ATTR1++2HeartLung carcinoma, atrial fibrillation85/M+ATTR1++3HeartTrauma84/M+ATTR1++4HeartHepatocellular carcinoma, hypertension80/M+ATTR1++5HeartHypertensive heart disease90/M+ATTR1++6HeartMyocardial amyloidosis, atrial fibrillation96/M+ATTR1++7HeartChronic pneumonia85/F+ATTR1++8EsophagusLung and prostate carcinoma, rheumatoid arthritis77/M+AA2, 3++8KidneyLung and prostate carcinoma, rheumatoid arthritis77/M+AA2, 3++9HeartHypertensive heart disease82/M+ATTR1++10HeartCardiomyopathy85/M+ATTR1++11HeartPrimary amyloidosis84/F+AL2++12HeartMultiple myeloma75/F+AL2++12TongueMultiple myeloma75/F+AL2++13HeartIleal adenocarcinoma75/F+ATTR1++14KidneyMonoclonal gammopathy73/F+AL2++15Seminal vesicleLung carcinoma73/M+AL2++16HeartCardiomyopathy88/M+ATTR1++17HeartAmyloidosis, myocardial infarction95/M+ATTR1++18HeartCoronary heart disease, suspected amyloidosis76/M+ATTR1++19StomachAbdominal pain71/M+AL2++20HeartSuspected amyloidosis79/M+ATTR1++21HeartTrauma17/F−−−−−21PancreasTrauma17/F−−−−−21SpleenTrauma17/F−−−−−21KidneyTrauma17/F−−−−−M, male; F, female.* Positive or negative staining are represented with + or −, respectively.† Amyloid subtype according to immunohistochemistry (IHC).‡ The numbers 1, 2, or 3 refer to the spectral classification of PTAA tingibility. Open table in a new tab Table 2Clinical Description and Tinctorial Properties of Additional SamplesPatientOrganClinical informationAge/SexCongo Red*Positive or negative staining are represented with + or −, respectively.IHC†Amyloid subtype according to immunohistochemistry (IHC).PTAA group‡The numbers 1, 2, or 3 refer to the spectral classification of PTAA tingibility.8HeartLung and prostate carcinoma, rheumatoid arthritis77/M+AA2, 38LungLung and prostate carcinoma, rheumatoid arthritis77/M+++AA2, 38LiverLung and prostate carcinoma, rheumatoid arthritis77/M++AA2, 38ColonLung and prostate carcinoma, rheumatoid arthritis77/M++AA2, 38ProstateLung and prostate carcinoma, rheumatoid arthritis77/M++AA2, 38ThyroidLung and prostate carcinoma, rheumatoid arthritis77/M+++AA2, 38Adrenal glandLung and prostate carcinoma, rheumatoid arthritis77/M+++AA2, 38ParathyroidLung and prostate carcinoma, rheumatoid arthritis77/M+++AA2, 312LungMultiple myeloma75/F+++AL222Lymph nodeLymphadenopathy49/F+++AA323KidneyProteinuria, suspected amyloidosis36/F+AA324KidneyProteinuria17/M+AA325Small intestineDiarrhea, nephrotic syndrome38/M+++AA325StomachDiarrhea, nephrotic syndrome38/M+++AA326DuodenumDuodenal ulcer53/M++AL227DuodenumSuspected amyloid47/M++AL228Sigma colonAL amyloidosis39/F+AL229HeartSuspected amyloidosis74/M+++ATTR130HeartSuspected cardiomyopathy62/M++AL231HeartHeart failure, monoclonal gammopathy80/M++ATTR132HeartCoronary heart disease, suspected AL amyloidosis87/M++ATTR133HeartProteinuria, diarrhea, arrhythmia77/M+++AL234HeartMultiple myeloma61/F++AL235HeartMultiple myeloma60/F+++ATTR136HeartMultiple myeloma68/M++AL237HeartMultiple myeloma90/M+AL238HeartMultiple myeloma, nephrotic syndrome54/M++AL239Joint capsuleFibrosis67/M+++AA340Soft tissueAmyloidosis71/M++AL241Joint capsuleArthritis70/M++AL242ConjunctivaSmoldering lymphoma with AL amyloidosis63/M+++AL243Soft tissueSuspected Ganglion, amyloid64/M+++AL244HeartMultiple myeloma, heart disease57/M−NegNeg45HeartMultiple myeloma, heart failure31/M−NegNeg46Joint capsuleGonarthrosis74/F−NegNeg47LungChronic obstructive pulmonary disease64/F−NegNeg48LungSuspected malignancy62/F−NegNeg49StomachFabry's disease17/M−NegNeg49DuodenumFabry's disease17/M−NegNegM, male; F, female.* Positive or negative staining are represented with + or −, respectively.† Amyloid subtype according to immunohistochemistry (IHC).‡ The numbers 1, 2, or 3 refer to the spectral classification of PTAA tingibility. Open table in a new tab M, male; F, female. M, male; F, female. When performing immunostains with antibodies to AL, we detected weak signals in all amyloid deposits. This is consistent with the reports that Ig λ-light chains frequently contaminates amyloid deposits of all types and can give rise to misinterpretations.13Picken MM New insights into systemic amyloidosis: the importance of diagnosis of specific type.Curr Opin Nephrol Hypertens. 2007; 16: 196-203Crossref PubMed Scopus (96) Google Scholar, 20Lachmann HJ Booth DR Booth SE Bybee A Gilbertson JA Gillmore JD Pepys MB Hawkins PN Misdiagnosis of hereditary amyloidosis as AL (primary) amyloidosis.N Engl J Med. 2002; 346: 1786-1791Crossref PubMed Scopus (535) Google Scholar, 21Novak L Cook WJ Herrera GA Sanders PW AL-amyloidosis is underdiagnosed in renal biopsies.Nephrol Dial Transplant. 2004; 19: 3050-3053Crossref PubMed Scopus (67) Google Scholar, 22Kebbel A Röcken C Immunohistochemical classification of amyloid in surgical pathology revisited.Am J Surg Pathol. 2006; 30: 673-683Crossref PubMed Scopus (120) Google Scholar, 23Satoskar AA Burdge K Cowden DJ Nadasdy GM Hebert LA Nadasdy T Typing of amyloidosis in renal biopsies—diagnostic pitfalls.Arch Pathol Lab Med. 2007; 131: 917-922PubMed Google Scholar Therefore, AL-positive samples were classified as AL amyloid only if they were negative for all other immunohistochemical amyloid stains (AA and ATTR). All other sections were initially stained with the AA antibody. If the samples were negative for AA, immunohistochemistry for ATTR and/or AL (m-κ, p-κ, m-λ, and p-λ) was performed. Of the 42 patients with amyloidosis studied here, immunohistochemical typing determined that 18 were ATTR, 18 were AL, and 6 were AA (Tables 1 and 2). The plausibility of immunohistochemical typing was corroborated by the evaluation of clinical diagnoses. For negative controls, we used 11 tissue samples from three patients with neoplastic diseases (multiple myeloma without amyloidosis; lung cancer), two patients with chronic inflammatory disorders (chronic obstructive pulmonary disease; gonarthrosis), one patient with lysosomal storage disease (Fabry's disease), and one young patient who died as a result of trauma and was devoid of any systemic or organ-specific amyloid (Tables 1 and 2). In a first series of experiments, the sensitivity and selectivity of LCPs were tested with an amyloid TMA containing samples from 20 amyloidosis patients and one negative control. PTAA, tPTAA, and p-FTAA (Figure 1, A–C) identified all amyloid deposits that were stained by antibodies and Congo Red, whereas none of the negative control samples showed any LCP signal (Figure 2, A–D). Amyloid deposits stained with PTAA or tPTAA emitted yellow-red fluorescence, whereas p-FTAA emitted extremely intense green-yellow fluorescence.Figure 2Immunohistochemical stains, Congo Red (CR) polarization microscopy, and LCP histochemistry of selected TMA spots. Each panel (A–D) depicts serial TMA sections stained with H&E, antibodies to the relevant amyloid constituents (ATTR, AA, AL, B2M), and LCPs as indicated. The middle columns depict original fluorescent micrographs, whereas the panels on the right represent p
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