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Application of confocal laser scanning microscopy for the diagnosis of amyloidosis

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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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References

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

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

    CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. 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 62:768–773

    CAS  PubMed  Google Scholar 

  8. 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 13:1–37

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  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 9:108–114

    CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  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 88:1024–1037

    Article  CAS  PubMed  Google Scholar 

  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 48:1067–1077

    Article  CAS  PubMed  Google Scholar 

  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):1844–1847

    Article  CAS  PubMed  Google Scholar 

  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):273–280

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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 9:863–869

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  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):590–593

    Article  Google Scholar 

  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 31:300–306

    Article  PubMed  Google Scholar 

  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 122:173–177

    Article  PubMed  Google Scholar 

  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 105:41–48

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  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 238:95–101

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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 88:232–242

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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 40:86–91

    Article  PubMed  Google Scholar 

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Acknowledgments

The authors thank Elisabetta Baliello, Alessandra Dubrovich, Luca Braghetto, Daniele Iannazzone and Giulia Fregonese for skillful technical assistance.

Authors’ contributions

CC, MF and AA performed the research; MD and MV performed ME experiments; AA, CC and MF designed the research study; AA contributed essential reagents and tools; CC, MF and ACF analysed the data; CC, MF and AA wrote the paper; AA, MV, FA and GT reviewed the paper.

All authors were involved in writing the manuscript and approving the final submitted version.

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Authors and Affiliations

  1. Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Via Gabelli 61, 35100, Padova, Italy

    Chiara Castellani, Marny Fedrigo, Anna Chiara Frigo, Mila Della Barbera, Gaetano Thiene, Marialuisa Valente & Annalisa Angelini

  2. Department of Clinical and Experimental Medicine, University of Padua, Padua, Italy

    Fausto Adami

Authors
  1. Chiara Castellani
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  2. Marny Fedrigo
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  3. Anna Chiara Frigo
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  4. Mila Della Barbera
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  5. Gaetano Thiene
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  6. Marialuisa Valente
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  7. Fausto Adami
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  8. Annalisa Angelini
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Correspondence to Annalisa Angelini.

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The authors declare that they have no conflict of interest.

University fundings

This work was supported by research grants from the Veneto Region, RF-VEN303/09, and from the University of Padua: CPDR099073, CPDA108809, 60A07-5074, 60A07-8587 and GRIC13IH1H.

Additional information

Chiara Castellani and Marny Fedrigo contributed equally to this work.

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Castellani, C., Fedrigo, M., Frigo, A.C. et al. Application of confocal laser scanning microscopy for the diagnosis of amyloidosis. Virchows Arch 470, 455–463 (2017). https://doi.org/10.1007/s00428-017-2081-7

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  • DOI: https://doi.org/10.1007/s00428-017-2081-7

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