Amyloidosis: Typing by immunohistochemistry and mass
spectrometry
Session
Nephropathology: Fibrillar glomerulopathies
Author
Dr. Linke, Reinhold
Abstract
1. Diagnosis of amyloid and amyloidosis: The term amyloidosis
encompasses a multitude of different amyloid diseases which are caused and
characterized by the presence of amyloid which is diagnosed on paraffin tissue
sections of affected organs using the alkaline Congo red staining method of
Puchtler et al. (cited in 1). The amyloid is microscopically recognizable by
specific pink staining in bright light, by green birefringence in polarized light
and by its orange-red Congo red fluorescence. Electron microscopically, these
deposits contain amyloid fibrils as their major constituent. The most decisive
pitfalls in diagnosing amyloid in tissue sections represent the sampling errors
and an inappropriate application of Puchtler's staining method. Decisive is in
particular the experience of the evaluator, since diagnosing amyloid is not
trivial and requires an experienced evaluator (1, 2). 2. Chemical diversity of
amyloids and corresponding, clinical amyloid diseases: Amyloid looks very
similar under histological and electron microscopic observation. However, in
the diverse clinical syndromes, amyloid is biochemically diverse as well. Each
amyloid protein defines a specific class of amyloidosis. Today, approximately
30 different amyloid classes are known. These classes of amyloid and
corresponding amyloid diseases (amyloidoses) are diagnosed in patients
through biopsies. Furthermore, some of the amyloid classes can display a
variety of clinical and genetic variants, thereby resulting in additional diversity
within one amyloid class amounting to a plethora of more than 500 different
individual amyloid diseases which need to be diffentiated in order to apply the
respective therapy (1 - 8). 3. Pathogenesis and therapy:
Each of the
pathogenetically different amyloid types needs a different amyloid-type specific
therapy. This necessarily implies that the amyloid protein must be identified
precisely in a routine clinicopathological practice in every patient. 4.
Amyloid classification: The classification of all amyloids identified is most
efficiently performed by immunohistochemistry, which is the most widely used
routine-method for classification of amyloid (4). For reliable
immunohistochemical classification we use homologous amyloid-type
antibodies (Table 1) in association with the PAP- or the ABC-method (1, 3-5).
Homologous amyloid-type antibodies were produced by immunogens isolated
from amyloid-loaden human organs (4). Another method for amyloid typing is
mass spectrometry (6,7).
5. Prevalence of amyloid classes: The results of
amyloid typing on tissue samples submitted to us by physicians in various
medical specialities can be read as in Table 2. The overall results are as
follows: We classified correctly 119 amyloid prototypes (with known amyloid) in
100% of the cases. The unknown amyloids of 581 patients submitted to us for
routine typing were classified correctly in 97.8%. The prevalence of different
types of amyloid is shown in Table 3. 6. Comparison of
immunohistochemistry and mass spectrometry: Three scientists organized a
collaborative project in order to compare amyloid typing from the same tissue
block with both methods immunohistochemistry and mass spectroscopy in a
blinded fashion called Ringstudy I. It lasted from 2002 till 2009 and was
completed in three parts with breaking the code three times from 2004 till 2009
with similar results in all three parts. The results were surprising: Comparisons
of immunohistochemistry and mass spectrometric findings on 91 different
amyloids were correct in 43(47.3%) by mass spectrometry and 87(95.6%) by
immunohistochemistry. This difference in sensitivity is statistically significant,
thus indicating that IHC is by far more sensitive than MS, while the different
specificities were statistically not significant. 7. Comparison of
immunohistochemistry and mass spectroscopy with antecedent micro
dissection (7): This study called Ringstudy II was initiated in February 2010
as a blinded study between two scientists using the same samples as in
Ringstudy I. It was concluded in August 2011 by breaking the code and is
ready to be published. 8. Conclusions: Our immunohistochemical typing
in routine use was reliable in 97,8 % of amyloids with homologous amyloidtype antibodies. Due to its very high sensitivity immunohistochemistry can type
minute amounts of amyloid in very small biopsies which have been shown to
be inaccessible for mass spectrometry. In cases where mass spectrometry
fails to type amyloid, the samples can be submitted for immunohistochemical
typing and, by doing so, spare the patient another biopsy and possibly its
necessity at a later date. However, when immunohistochemistry was unable to
type a particular amyloid, mass spectrometry has been seen to be successful
in some cases.
References
1. Linke RP. Congo red staining of amyloid.
Improvements and practical guide for a more precise diagnosis of amyloid and
the different amyloidoses. In: Uversky VN and Fink AL, eds. Protein
misfolding, aggregation and conformational diseases. Protein Reviews,
Springer. 2006;Volume 4, Chapter 11.1:239-276.
2. Picken, MM,
Westermark P. Amyloid detection and typing: summary of current practice and
recommendations of the group. Amyloid 2011:18 (Suppl. 1); 48-50. 3.
Linke, R.P., Oos, R., Wiegel, N.M., Nathrath, W.B.J. Classification of
amyloidosis: Misdiagnosing by way of incomplete immunohistochemistry and
how to prevent it. Acta Histochem. 2006;108:197-208.
4. Linke RP. On
typing amyloidosis using immunohistochemisty. Detailed illustrations, review
and a note on mass spectrometry. Progr Histochem Cytochem 2012; 47: 61132. 5. Linke RP. Routine use of amyloid typing on formalin-fixed paraffin
sections from 626 patients by immunohistochemistry. In: Amyloid and Related
Disorders: Surgical Pathology and Clinical Correlations, Current Clinical
Pathology. Picken MM, et al, (eds.) Chapter 17, pp 219-229; Springer Science
& Business Media. 2012.
6. Murphy CL, Wang S, Williams T et al.
Characterization of systemic amyloid deposits by mass spectrometry. Methods
Enzymol. 2006; 412: 48-62. 7. Vrana JA, Gamez JD, Madden BJ, Theis
JD, Bergen HR 3rd, Dogan A. Classification of amyloidosis by laser micro
dissection and mass spectrometry based proteomic analysis in clinical biopsy
specimens. Blood. 2009;114:4957-4959.
8. Linke RP, Westermark P,
Solomon A. Classification of amyloidosis, comparison of two leading routine
methods: immunohistochemistry and mass spectrometry, procedure and first
results. In: Hazenberg BP, Bijzet J, van Gameren II, Limburg PC, Gruys E, and
van Rijswijk MH, editors. XIIIth International Symposium on Amyloidosis.
Groningen: UMCG, 2013: [in press].