Typing of Amyloid for Routine Use on Formalin-Fixed Paraffin Sections of 626 Patients by Applying Amyloid type-Specific Immunohistochemistry: A Review
After establishing a diagnosis of amyloid by the Congo red procedure (see Chap. 14), it is now considered mandatory, for therapeutic reasons, to identify the chemical nature (i.e., “type”) of the amyloidotic protein causing the disease. In this review, it is shown how adequate and reliable immunohistochemical typing of amyloid can be achieved using formalin-fixed, paraffin-embedded tissue sections and a panel of amyloid type-specific antibodies. In this technique, antibodies are raised against the amyloidotic proteins themselves and, thereby, avoid the pitfalls and deficiencies associated with antibodies prepared against native, soluble, precursor proteins, which often lead to suboptimal results, typically reported as “unreliable immunohistochemistry.” These amyloid type-specific antibodies, prepared and evaluated with appropriate controls, therefore constitute the key to high-quality immunohistochemical amyloid typing.
In preparing these antibodies as reagents, only a strong and consistent reaction against a single amyloid protein type is considered diagnostic, and needs to be distinguished from other reactions, which are weaker and inconsistent, and therefore deemed nonspecific. These latter types of reactions are predictable considering that amyloid deposits represent a complex composite of many different constituents that are present in addition to the amyloid protein itself. In fact, these other constituents have hindered amyloid typing efforts, regardless of the method used, for decades.
The sensitivity and specificity of immunohistochemistry (IHC) can be further increased by double staining, using Congo red as the first step, followed by an immunohistochemical (CRIC) stain as an overlay. This overlay stain allows concomitant evaluation of the spatial relationship between Congo red-positive and immune stain-positive areas. All 19 different types of amyloid can be diagnosed by using a set of different amyloid type-specific antibodies and comparing their reactivities. Thus, the amyloid protein type is diagnosed based on the most dominant and consistent stain, while the weaker, nondiagnostic stains exclude all other amyloid types; this type of “comparative” approach to the interpretation of IHC stains thus provides support for its reliability through the inclusion of internal controls. In the author’s experience, based on more than 626 submitted samples, and using this comparative approach, the sensitivity of IHC is >97 %, and the specificity even higher; there were no major cross-reacting stains. Other laboratories have also achieved similar reliable results, on a routine basis, using amyloid-specific antibodies. Finally, the reliability of the typing results obtained usingamyoid type-specific antibodies was confirmed by mass spectrometric analysis of isolated amyloid proteins in two collaborative international blind-comparison studies.
KeywordsAmyloid type prototypes Formalin-fixed paraffin sections Amyloid type-specific Immunohistochemistry (IHC) Double staining: Congo red with immunohistochemical overlay (CRIC) Amyloid typing Pitfalls in typing of amyloid Isolated amyloid fibrils Native proteins Nonnative proteins Amyloidotic proteins Conformational change Variable and unpredictable immunoreactivity Sensitivity Specificity
This work was only possible as a result of contributions by many colleagues over several decades. Full acknowledgement of some of these contributions can be found in an in-depth review . Here, I would like to thank Prof. Dr. R. Huber, director emeritus of the Max-Planck-Institute of Biochemistry in Martinsried, who provided laboratory space, and supported some of the technicians and coworkers involved in the laboratory work. They include Mrs. A. Rail, Mrs. A. Kerling, Mrs. R. Oos, Mrs. A. Meinel, and Dr. N. Wiegel. For secretarial work, I thank Mrs. A. Feix, Martinsried, Germany, and for the artwork Ms. A. K. M. Linke, Essen/Germany.
- 4.Linke RP. Congo red staining of amyloid. Improvements and practical guide for a more precise diagnosis of amyloid and the different amyloidosis. Chapter 11.1. In: Uversky VN, Fink AL, editors. Protein misfolding, aggregation and conformational diseases, Protein Reviews, vol. 4. New York: Springer; 2006. p. 239–76.CrossRefGoogle Scholar
- 5.Schroeder R, Deckert M, Linke RP. Novel isolated cerebral ALλ (lambda) amyloid angiopathy with widespread subcortical distribution and leukoencephalopathy due to atypical monoclonal plasma cell proliferation, and terminal systemic gammopathy. J Neuropathol Exp Neurol. 2009;68(3):286–99.CrossRefGoogle Scholar
- 11.Linke RP, Joswig R, Murphy CL, et al. Seminogelin I is the amyloidogenic protein in senile seminal vesicles. In: Grateau G, Kyle RA, Skinner M, editors. Amyloid and amyloidosis. Boca Raton, FL: CRC Press; 2004. p. 471–3.Google Scholar
- 13.Linke RP. On typing of amyloidosis using immunohistochemstry. Detailed illustrations, review and a note on mass spectrometry. Progr Histochem Cytochem. 2012;47:61–132.Google Scholar