Amyloid class
No. of patients
% of total
ALλ
271
43.3
ALκ
118
18.8
(AL, sum)
(389)
(62.1)
ATTR
93
14.9
AA
80
12.8
AFib
14
2.2
Aβ2M
11
1.8
ALys
6
1.0
AApoAI
5
0.8
Aβ
4
0.6
APrP
2
0.3
ACys
2
0.3
AHγ
2
0.3
AGel
2
0.3
AApoAII
1
0.2
SAA4
1
0.2
Unknown
14
2.2
Sum total
626
100
The IHC technique used for amyloid typing by our laboratory (and also by others) is the unlabeled IHC technique of Sternberger (cited in ref. [4]), in which a peroxidase–anti-peroxidase (PAP) complex is applied as the final amplification system, or its more sensitive variant—the ABC technique which uses a biotin–avidin complex for amplification. Before beginning the IHC staining procedure, one section must be examined in order to verify the presence of amyloid in the submitted biopsy. When smaller amounts of amyloid are present, or if it is suspected that a sampling error might occur (see “sampling error” in Chap. 14), approximately 15 sections are prestained for 10–20 s with Congo red (CR) using a modified Puchtler method [4]. This procedure incorporates the absolute minimum time necessary for staining so that the CR staining will show up only by Congo red fluorescence (CRF), a more sensitive technique than the classical CR method employing bright light [4, 13]. This brief prestaining treatment therefore ensures that the CR staining will not interfere with the IHC procedure. Amyloid that fluoresces by CRF needs to be verified by polarization microscopy, when the pathognomonic green birefringence (GB) should be seen [4]. Sections that show the presence of amyloid are then chosen for IHC staining. Whenever the procedure needs to be changed, consequent to the inclusion of new solutions, other reagents, new personnel, etc., we routinely include seven positive controls (one for each of the five major amyloid classes) in order to make sure that the applied technique will be of a similar high standard and that the results will be comparable to former IHC stains. Positive controls, after evaluation, can be destained (using 80 % acetic acid dropped onto the section for 2 min) and reused for IHC. In our laboratory, we routinely reuse positive control slides up to approximately six times [4]. The staining procedure utilizes routine methods with AEC (3-amino-ethyl carbazol) as the chromogen, followed by a weak counterstain with Mayer’s hemalum, and embedding in Kaiser’s glycerin jelly [4].
Evaluation of Immunohistochemical Reactivities
The reading of IHC-stained slides is performed without any prior knowledge of either laboratory data or clinical picture. Positive standard slides are first evaluated in order to provide assurance that the “unknown” slides of submitted tissue sections have been stained properly with the set of antibodies employed. The second stage of the procedure is then to read the ten stained slides and evaluate their IHC reactivities. This requires some experience since amyloid represents a complex mixture of many proteins and other constituents [8–10, 13] that will show various reactivities. The latter need to be categorized by distinguishing strong uniform and consistent IHC reactions from the more inconsistent ones in order to separate the specific and diagnostic from the nonspecific reactivities.
This is shown in Figs. 18.1, 18.2, 18.3, and 18.4. The amyloid typing is illustrated here using the four most prevalent amyloid types: AA, ALλ (lambda), ALκ (kappa), and ATTR (more types have been illustrated in [10, 13]). CRF was used to identify the presence of amyloid in each of the four presented cases since it is the most sensitive method for confirming the presence of amyloid (see Chap. 14). Amyloid is shown by CRF in the “a-row” of all cases in Figs. 18.1, 18.2, 18.3, and 18.4. IHC typing of amyloid was performed on adjacent sections, to which the above-described panel of amyloid type-specific antibodies was applied. All three forms of illumination used for identifying amyloid (triple illumination) were applied to all amyloid types, as exemplified in the ATTR case in Fig. 18.4b.
Fig. 18.1
Immunohistochemical classification using amyloid type-specific antibodies. Figures 18.1, 18.2, 18.3, and 18.4 show the classification of four different amyloid types as AA, ALλ, ALκ, and ATTR, respectively. Here, stains using four antibodies are presented [5, 8, 10] to illustrate the principle of comparative amyloid typing. The results of CRF are shown in row (a), the reaction with anti-AA antibodies across the different amyloid types in row (b), the reactivities against ALλ, ALκ, and ATTR are shown in rows (c, d, and e), respectively. The diagnostic reaction is always the strongest and most consistent (see text)
Fig. 18.2
Immunohistochemical classification using amyloid type-specific antibodies. Figures 18.1, 18.2, 18.3, and 18.4 show the classification of four different amyloid types as AA, ALλ, ALκ, and ATTR, respectively. Here, stains using four antibodies are presented [5, 8, 10] to illustrate the principle of comparative amyloid typing. The results of CRF are shown in row (a), the reaction with anti-AA antibodies across the different amyloid types in row (b), the reactivities against ALλ, ALκ, and ATTR are shown in rows (c, d, and e), respectively. The diagnostic reaction is always the strongest and most consistent (see text)
Fig. 18.3
Immunohistochemical classification using amyloid type-specific antibodies. Figures 18.1, 18.2, 18.3, and 18.4 show the classification of four different amyloid types as AA, ALλ, ALκ, and ATTR, respectively. Here, stains using four antibodies are presented [5, 8, 10] to illustrate the principle of comparative amyloid typing. The results of CRF are shown in row (a), the reaction with anti-AA antibodies across the different amyloid types in row (b), the reactivities against ALλ, ALκ, and ATTR are shown in rows (c, d, and e) respectively. The diagnostic reaction is always the strongest and most consistent (see text)
Fig. 18.4
Immunohistochemical classification using amyloid type-specific antibodies. Figures 18.1, 18.2, 18.3, and 18.4 show the classification of four different amyloid types as AA, ALλ, ALκ, and ATTR, respectively. Here, stains using four antibodies are presented [5, 8, 10] to illustrate the principle of comparative amyloid typing. The results of CRF are shown in row (a), the reaction with anti-AA antibodies across the different amyloid types in row (b), the reactivities against ALλ, ALκ, and ATTR are shown in rows (c, d, and e) respectively. The diagnostic reaction is always the strongest and most consistent (see text)
In Fig. 18.1a–e, characteristic reactivities are shown for renal autopsy tissue with glomerular AA amyloid (in a case of Muckle-Wells syndrome, see ref. in [4]) as identified by CRF (Fig. 18.1a) and a very consistent and strongly congruent diagnostic reaction with anti-AA only (Fig. 18.1b, between arrows) while anti-ALκ and anti-ATTR (Fig. 18.1d, e) were nonreactive. The anti-ALλ (lambda) antibody (Fig. 18.1c) showed a distinct reactivity; however, this was weak in comparison to the reactivity shown in Fig. 18.1b which, being the strongest, was considered diagnostic of the amyloid type. Please note that light chain amyloid antibodies also react strongly with amyloid-free tissue structures such as tubular cells (Fig. 18.1c, d).
In Fig. 18.2a–e, IHC identified ALλ (lambda) amyloid as the type; here, amyloid is seen only in the cardiac vessels in a patient with cardiac decompensation and MGUS (free λ-L 343 mg/l serum). The cardiac functional decompensation resulted from severe narrowing of the vessels by amyloid (Fig. 18.2b–e, between arrows) leading to functional impairment, despite the cardiac muscle being virtually free of amyloid. The amyloid was barely stained with Congo red (not shown) but was visible with CRF illumination (Fig. 18.2a). Diagnosis of the amyloid type was based on identification of a single, consistent, and very strong IHC reaction (corresponding to the location of amyloid deposits by CRF) with anti-ALλ (lambda) only (Fig. 18.2c). In contrast, the anti-ALκ (kappa) (Fig. 18.2d) stain was less strong and less uniform. There was almost no reactivity with anti-AA (Fig. 18.2b) and anti-ATTR (Fig. 18.2e) except for a few reactive and inconsistent spots in the latter. Please note that considerable reactivity is present with anti-ATTR in cardiomyocytes, which does not, however, correspond in location to the areas positive for amyloid by CRF and, therefore, is not specific for amyloid.
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