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01-04-2017 | Original Article

Application of confocal laser scanning microscopy for the diagnosis of amyloidosis

Authors: Chiara Castellani, Marny Fedrigo, Anna Chiara Frigo, Mila Della Barbera, Gaetano Thiene, Marialuisa Valente, Fausto Adami, Annalisa Angelini

Published in: Virchows Archiv | Issue 4/2017

Abstract

We analysed specificity and sensitivity of confocal laser microscopy (CLSM) on tissue sections for a diagnosis of amyloidosis, in an attempt to reduce technical errors and better standardise pathological diagnosis. We first set up a protocol for the use of CLSM on this type of specimen, using a group of 20 amyloid negative and 20 positive samples. Of all specimens, 2, 4 and 8-μm sections were cut. Sections were stained with Congo red (CR) and thioflavin-T (ThT) and observed by cross-polarised light microscopy (CR-PL), epifluorescence microscopy (CRF-epiFM and ThT-epiFM) and CLSM (CRF-CLSM and ThT-CLSM). To validate the method in a diagnostic setting, we examined tissue samples from 116 consecutive patients with clinical suspicion of amyloidosis, selected from the period 2005 to 2014 from the database of the Pathology Unit of the University of Padua. The results were compared with those of transmission electron microscopy (TEM), which we consider as reference. We found that with CRF-CLSM, the false negative rate was reduced from 17 to 5%, while the sensitivity of detection increased to 12%. The results were in complete agreement with those of TEM ThT-CLSM; both sensitivity and specificity were 100%. Finally, ThT-CLSM results did not vary with section thickness, and small amounts of amyloid could even be detected in 2-μm sections. In conclusion, we found ThT-CLSM to be more sensitive as a screening method for amyloidosis than CR and ThT epifluorescence optical imaging. The method was easier to standardise, provided images with better resolution and resulted in more consistent pathologist diagnoses.

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Westermark P, Benson MD, Buxbaum JN et al (2007) A primer of amyloid nomenclature. Amyloid 14:179–183CrossRefPubMed

Sipe JD, Benson MD, Buxbaum JN et al (2014) Nomenclature 2014: amyloid fibril proteins and clinical classification of the amyloidosis. Amyloid 21:221–224CrossRefPubMed

Kholova I, Niessen HW (2005) Amyloid in cardiovascular system: a review. JClinPathol 58:125–133

Hawkins PN (1995) Amyloidosis. Blood Rev 9:135–142CrossRefPubMed

Le Vine H (1995) Thioflavine T interaction with amyloid β-sheet structures. Amyloid 2:1–6CrossRef

Naiki H, Higuchi K, Hosokawa M, Takeda T (1989) Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem 177:244–249CrossRefPubMed

Naiki H, Higuchi K, Matsushima K et al (1990) Fluorometric examination of tissue amyloid fibrils in murine senile amyloidosis: use of the fluorescent indicator, thioflavine T. Lab Investig 62:768–773PubMed

Glenner GG (1981) The bases of the staining of amyloid fibers: their physico-chemical nature and the mechanism of their dye–substrate interaction. Prog Histochem Cytochem 13:1–37CrossRefPubMed

Rocken C, Schwotzer EB, Linke P, Saeger W (1996) The classification of amyloid deposits in clinicopathological practice. Histopathology 29:325–335CrossRefPubMed

10.

Linke RP (2012) On typing amyloidosis using immunohistochemistry. Detailed illustrations, review and a note on mass spectrometry. Prog Histochem Cytochem 47(2):61–132CrossRefPubMed

11.

Arbustini E, Verga L, Concardi M, Palladini G, Obici L, Merlini G (2002) Electron and immuno-electron microscopy of abdominal fat identifies and characterizes amyloid fibrils in suspected cardiac amyloidosis. Amyloid 9:108–114PubMed

12.

Fernández de Larrea C, Verga L, Morbini P et al (2015) A practical approach to the diagnosis of systemic amyloidoses. Blood 125:2239–2244CrossRefPubMed

13.

Rodriguez FJ, Gamez JD, Vrana JA et al (2008) Immunoglobulin derived depositions in the nervous system: novel mass spectrometry application for protein characterization in formalin-fixed tissues. Lab Investig 88:1024–1037CrossRefPubMed

14.

Theis JD, Dasari S, Vrana JA, Kurtin PJ, Dogan A (2013) Shotgun-proteomics-based clinical testing for diagnosis and classification of amyloidosis. J Mass Spectrom 48:1067–1077CrossRefPubMed

15.

Brambilla F, Lavatelli F, Di Silvestre D, Valentini V, Rossi R, Palladini G, Obici L, Verga L, Mauri P, Merlini G (2012) Reliable typing of systemic amyloidosis through proteomic analysis of subcutaneous adipose tissue. Blood 119(8):1844–1847CrossRefPubMed

16.

Sethi S, Vrana JA, Theis JD, Dogan A (2013) Mass spectrometry based proteomics in the diagnosis of kidney disease. Curr Opin Nephrol Hypertens 22(3):273–280CrossRefPubMed

17.

Picken MM (2015) Proteomics and mass spectrometry in the diagnosis of renal amyloidosis. Clin Kidney J 8(6):665–672CrossRefPubMedPubMedCentral

18.

Linke RP (2000) Highly sensitive diagnosis of amyloid and various amyloid syndromes using Congo red fluorescence. Virchows Arch 436:439–448CrossRefPubMed

19.

Linke RP, Gärtner HV, Michels H (1995) High sensitive diagnosis of AA-amyloidosis using Congo red and immunohistochemistry detects missed amyloid deposits. J Histochem Cytochem 9:863–869CrossRef

20.

Michels H, Linke RP (1998) Clinical benefits of diagnosing incipient AA amyloidosis in pediatric rheumatic diseases as estimated from a retrospective study. Amyloid 5:200–207CrossRefPubMed

21.

Elghetany MT, Saleem A, Barr K (1989) The Congo red stain revisited. Ann Clin Lab Sci 19:190–195PubMed

22.

Marcus A, Sadimin E, Richardson M, Goodell L, Fyfe B (2012) Fluorescence microscopy is superior to polarized microscopy for detecting amyloid deposits in Congo red–stained trephine bone marrow biopsy specimens. Am J Clin Pathol 38(4):590–593CrossRef

23.

Giorgadze TA, Shiina N, Baloch ZW, Tomaszewski JE, Gupta PR (2004) Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy. Diagn Cytopathol 31:300–306CrossRefPubMed

24.

Turillazzi E, Karch SB, Neri M, Pomara C, Riezzo I, Fineschi V (2008) Confocal laser scanning microscopy. Using new technology to answer old questions in forensic investigations. Int J Legal Med 122:173–177CrossRefPubMed

25.

White JG, Amos WB, Fordham M (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105:41–48CrossRefPubMed

26.

Amos WB, White JG (2003) How the confocal laser scanning microscope entered biological research. Biol Cell 95:335–342CrossRefPubMed

27.

Sanderson MJ, Smith I, Parker I, Bootman MD (2014) Fluorescence microscopy. Cold Spring Harb Protoc. doi:10.1101/pdb.top071795 PubMedPubMedCentral

28.

Falk RH (2005) Diagnosis and management of the cardiac amyloidoses. Circulation 112:2047–2060CrossRefPubMed

29.

Puchtler H, Sweat F, Levine M (1962) On the binding of Congo red by amyloid. J Histochem Cytochem 10:355–364CrossRef

30.

Michels J, Buntzow M (2010) Assessment of Congo red as a fluorescence marker for the exoskeleton of small crustaceans and the cuticle of polychaetes. J Microsc 238:95–101CrossRefPubMed

31.

Simel DL, Samsa GP, Matchar DB (1991) Likelihood ratios with confidence: sample size estimation for diagnostic test studies. J Clin Epidemiol 44:763–770CrossRefPubMed

32.

Westermark GT, Johnson KH, Westermark P (1999) Staining methods for identification of amyloid tissue. Methods Enzymol 309:3–25CrossRefPubMed

33.

Elghetany MT, Saleem A (1988) Methods for staining amyloid in tissues: a review. Stain Technol 63:201–212CrossRefPubMed

34.

Khurana R, Uversky VN, Nielsen L, Fink AL (2001) Is Congo red an amyloid-specific dye? J Biol Chem 276:22715–22721CrossRefPubMed

35.

Howie AJ, Brewer DB (2009) Optical properties of amyloid stained by Congo red: history and mechanisms. Micron 40:285–301CrossRefPubMed

36.

Howie AJ, Brewer DB, Howell D, Jones AP (2008) Physical basis of colors seen in Congo red-stained amyloid in polarized light. Lab Investig 88:232–242CrossRefPubMed

37.

Biancalana M, Koide S (2010) Molecular mechanism of thioflavin-T binding to amyloid fibrils. Biochim Biophys Acta 1804:1405–1412CrossRefPubMedPubMedCentral

38.

Khurana R, Coleman C, Ionescu-Zanetti C et al (2005) Mechanism of thioflavin T binding to amyloid fibrils. J Struct Biol 151:229–238CrossRefPubMed

39.

Vassar PS, Culling CF (1959) Fluorescent stains with special reference to amyloid and connective tissues. Arch Pathol 68:487–498PubMed

40.

Krebs MR, Bromley EH, Donald AM (2005) The binding of thioflavin-T to amyloid fibrils: localisation and implications. J Struct Biol 149:30–47CrossRefPubMed

41.

Scivetti M, Favia G, Fatone L, Maiorano E, Crincoli V (2016) Concomitant use of Congo red staining and confocal laser scanning microscopy to detect amyloidosis in oral biopsy: a clinicopathological study of 16 patients. Ultrastruct Pathol 40:86–91CrossRefPubMed

Title: Application of confocal laser scanning microscopy for the diagnosis of amyloidosis
Authors: Chiara Castellani
Marny Fedrigo
Anna Chiara Frigo
Mila Della Barbera
Gaetano Thiene
Marialuisa Valente
Fausto Adami
Annalisa Angelini
Publication date: 01-04-2017
Publisher: Springer Berlin Heidelberg
Published in: Virchows Archiv / Issue 4/2017
Print ISSN: 0945-6317
Electronic ISSN: 1432-2307
DOI: https://doi.org/10.1007/s00428-017-2081-7

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Application of confocal laser scanning microscopy for the diagnosis of amyloidosis

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