**4.7 Atypical amyloid findings**

Cases of intracellular amyloid deposition have been reported in few organs including cardiomyocytes, plasma cells, as well as the histiocytes and β cells of the pancreas [12–14]. Spheroid type (corpora amylacea-like) amyloid deposition is reported in pituitary adenoma, squamous cell carcinoma of the uterine cervix, and amyloidoma of the bone, jejunum, and colon [15–18]. One case of spheroid type amyloid deposition from our group in association with colon adenocarcinoma is identified [2] (**Figures 6** and **7**). Current hypothesis regarding spheroid type amyloid deposition is that during the process of amyloid removal by macrophages, amyloid is packed inside the macrophage, making spheroid formations that are extracted into the surrounding tissue [18].

## **5. Immunohistochemistry and immunoelectron microscopy**

While the Congo red stain positivity and birefringence are the gold standard of confirming amyloidosis, they do not tell what type of amyloid is deposited. Considering managements and clinical outcomes vary drastically according to the types, further studies to identify the causative protein are critical. Clinicopathologic correlation cannot substitute for amyloid typing.

Immunohistochemistry (IHC) is the most commonly utilized method for subtyping amyloidosis. IHC takes advantage of relatively specific binding properties of antibodies against different types of amyloid fibrils to illuminate the amyloid protein in tissue. A panel of antibodies for more common types can subtype the majority of amyloidosis cases. Such antibodies include those against ALλ, ALκ, AHγ, ATTR, Aβ2M, and AFib (fibrinogen). The method has been widely used due to low cost, ease of use, rapid turnaround time, and formalin-fixed paraffinembedded (FFPE) section compatibility.

However, there is one important pitfall in IHC. Because of heterogeneity of amyloid fibrils, nonspecific staining is common, and this potentially complicates the interpretation. For instance, the antibody against Aλ is notorious for nonspecific staining of amyloid other than Aλ. This diagnostic pitfall mandates the use of multiple comparative IHC stains to separate the true diagnostic positivity from the nonspecific reaction. In comparative IHC, subtyping of amyloid is determined by the specific amyloid with the strongest immunohistochemical reactivity.

**Figure 8.** *Amyloid fibrils diameter of approximately 7.5–10 nm non branched fibers in kidney, EM.*

Although not as commonly utilized as IHC, electron microscopy (EM) is a preferred method over Congo red birefringence or IHC in some institutions due to ambiguity of these stains in the interpretation. EM can confirm or rule out amyloidosis by visualizing amyloid fibrils in tissue as non-branching fibers with an average diameter of 7.5–10 nm. Because these fibers are considerably thicker than collagen fibers in EM, this technique can avoid diagnosing collagen fibers as amyloid fibrils, which is common in Congo red stain due to birefringence of collagen fibers in abdominal fat biopsy.

Morphologic patterns of EM have been described in amyloidosis affecting certain organs. Selective deposition in mesangial matrix and basement membrane and subepithelial "spikes" or "spicules" under podocyte foot processes are seen in glomerular amyloidosis (**Figure 8**). Amyloid deposition in tubular basement membrane, interstitial space, and vascular wall are observed in extraglomerular amyloid. However, such differences in distribution are not specific enough to indicate certain subtypes of amyloidosis.

Some authors may further utilize immunoelectron microscopy (IEM), in which immunogold stains—antibody probes conjugated with gold particles—for ALλ, ALκ, AA, and ATTR are used to subtype the amyloid fibril. These stains "decorate" the target amyloid fibers and can be seen as "beads" in the fibrillary matrix of amyloid. IEM can detect even small amounts of amyloid fibrils, at earlier stages of the disease. However, the processing deals with a very small piece of tissue and leads to a false-negative result due to the limited sampling, especially in cases where the amyloid deposits are focal. Another barrier is fixation, where architectural details are preserved by cross-linking, but at the same time loss of antigenicity may result from dehydration and embedding procedures. Therefore, alternative fixatives such as modified Karnovsky's solution rather than conventional glutaraldehyde with different protocols are used for IEM examination.

### **6. Molecular diagnosis**

Recent advances in MS-based proteomic analysis have revolutionized detecting and subtyping of amyloidosis. The analytic method has made it possible to detect and identify new kinds of amyloid fibrils as well as previously known ones in a given specimen without direct sequencing. One such example is ALECT2. As mentioned above, LECT2 is synthesized by the liver and released into the circulation

**71**

*Pathologic Findings of Amyloidosis: Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.84268*

sis and treated as such without MS-based analysis.

the proteins that are contained in a given specimen.

mandate the exact subtyping of amyloid.

variants is utilized.

type of amyloidosis.

**7. Conclusion**

and has uncertain physiologic roles in the cartilage and liver. It has been shown that ALECT2 is one of the major causes of kidney and liver amyloidosis after AL and AA amyloidosis, especially among Hispanics. This major amyloidosis may have been unrecognized due to a relatively indolent clinical course and limited ethnic distribution. Because serum LECT2 levels are not elevated, and no mutations are found in LECT2 gene so far, ALECT2 might have been misdiagnosed as AL or AA amyloido-

The MS-based proteomic analysis utilizes techniques like laser microdissection (LMD), high-performance liquid chromatography (HPLC), and a variety of computational database tools. Although earlier HPLC- and MS-based analysis suffered from lack of specificity due to heterogeneous nature of the specimen, LMD largely overcame such diagnostic inaccuracy. LMD deals with microscopic examination of the specimen, selection of a field of interest, and microdissection of the field using laser in an attempt to achieve pure amyloid plaques. The dissected specimen can be submitted for histochemistry, IHC, or MS analysis. For FFPE specimens, an extra step for protein release similar to antigen retrieval used in IHC is applied. The released proteins are treated with a proteolytic enzyme (most commonly trypsin), and the resultant digested peptide fragments are separated by HPLC and analyzed with MS. This analytic method is based on an assumption that each human protein has their unique tryptic fragmentation patterns, which serves as a "fingerprint" of the protein. The analysis involves a previously curated database on human proteins and a number of computational algorithms to predict the amino acid sequences of

Although the LMD- and MS-based proteomic analysis has demonstrated great sensitivity and specificity, they have one major pitfall. Because MS-based proteomic analysis heavily depends on human protein databases available in public, new polymorphisms or mutations may not be listed in the databases and, thus, cannot be identified using the technique. In such situations, a separate workflow to compare the newly identified mutations against previously known

Amyloidosis is characterized morphologically by amorphous deposition of amyloid within tissue. The deposition is caused by aggregation of misfolded protein. Any disruptive processes in protein homeostasis (proteinostasis) can cause such misfolding and aggregation. Although different species of amyloid protein have different organotropisms and physiochemical properties, they appear remarkably similar when deposited within the target tissue. Clinical signs and symptoms of different types are largely affected by the organ where the amyloid is deposited. However, different treatment modalities and clinical courses according to the type

The confirmation and subtyping of amyloidosis heavily depend on pathologic examination of abdominal fat, minor salivary gland, or target organs. The gold standard for confirmation of amyloidosis is Congophilia and birefringence. Additional modalities such as IHC, EM, and MS can help further subclassify the

Lately, new types of amyloidosis have been identified by MS. Atypical structure of amyloid continues to be found in various organs. Contrary to the conventional definition of amyloid, such as extracellular amorphous deposition, intracellular and spherical structure amyloids have been discovered. In addition, novel mutations of the same protein have been shown to confer totally different clinical implications.

#### *Pathologic Findings of Amyloidosis: Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.84268*

*Amyloid Diseases*

**Figure 8.**

subtypes of amyloidosis.

**6. Molecular diagnosis**

different protocols are used for IEM examination.

Although not as commonly utilized as IHC, electron microscopy (EM) is a preferred method over Congo red birefringence or IHC in some institutions due to ambiguity of these stains in the interpretation. EM can confirm or rule out amyloidosis by visualizing amyloid fibrils in tissue as non-branching fibers with an average diameter of 7.5–10 nm. Because these fibers are considerably thicker than collagen fibers in EM, this technique can avoid diagnosing collagen fibers as amyloid fibrils, which is common in Congo red stain due to birefringence of collagen fibers in abdominal fat biopsy. Morphologic patterns of EM have been described in amyloidosis affecting certain organs. Selective deposition in mesangial matrix and basement membrane and subepithelial "spikes" or "spicules" under podocyte foot processes are seen in glomerular amyloidosis (**Figure 8**). Amyloid deposition in tubular basement membrane, interstitial space, and vascular wall are observed in extraglomerular amyloid. However, such differences in distribution are not specific enough to indicate certain

*Amyloid fibrils diameter of approximately 7.5–10 nm non branched fibers in kidney, EM.*

Some authors may further utilize immunoelectron microscopy (IEM), in which immunogold stains—antibody probes conjugated with gold particles—for ALλ, ALκ, AA, and ATTR are used to subtype the amyloid fibril. These stains "decorate" the target amyloid fibers and can be seen as "beads" in the fibrillary matrix of amyloid. IEM can detect even small amounts of amyloid fibrils, at earlier stages of the disease. However, the processing deals with a very small piece of tissue and leads to a false-negative result due to the limited sampling, especially in cases where the amyloid deposits are focal. Another barrier is fixation, where architectural details are preserved by cross-linking, but at the same time loss of antigenicity may result from dehydration and embedding procedures. Therefore, alternative fixatives such as modified Karnovsky's solution rather than conventional glutaraldehyde with

Recent advances in MS-based proteomic analysis have revolutionized detecting and subtyping of amyloidosis. The analytic method has made it possible to detect and identify new kinds of amyloid fibrils as well as previously known ones in a given specimen without direct sequencing. One such example is ALECT2. As mentioned above, LECT2 is synthesized by the liver and released into the circulation

**70**

and has uncertain physiologic roles in the cartilage and liver. It has been shown that ALECT2 is one of the major causes of kidney and liver amyloidosis after AL and AA amyloidosis, especially among Hispanics. This major amyloidosis may have been unrecognized due to a relatively indolent clinical course and limited ethnic distribution. Because serum LECT2 levels are not elevated, and no mutations are found in LECT2 gene so far, ALECT2 might have been misdiagnosed as AL or AA amyloidosis and treated as such without MS-based analysis.

The MS-based proteomic analysis utilizes techniques like laser microdissection (LMD), high-performance liquid chromatography (HPLC), and a variety of computational database tools. Although earlier HPLC- and MS-based analysis suffered from lack of specificity due to heterogeneous nature of the specimen, LMD largely overcame such diagnostic inaccuracy. LMD deals with microscopic examination of the specimen, selection of a field of interest, and microdissection of the field using laser in an attempt to achieve pure amyloid plaques. The dissected specimen can be submitted for histochemistry, IHC, or MS analysis. For FFPE specimens, an extra step for protein release similar to antigen retrieval used in IHC is applied. The released proteins are treated with a proteolytic enzyme (most commonly trypsin), and the resultant digested peptide fragments are separated by HPLC and analyzed with MS. This analytic method is based on an assumption that each human protein has their unique tryptic fragmentation patterns, which serves as a "fingerprint" of the protein. The analysis involves a previously curated database on human proteins and a number of computational algorithms to predict the amino acid sequences of the proteins that are contained in a given specimen.

Although the LMD- and MS-based proteomic analysis has demonstrated great sensitivity and specificity, they have one major pitfall. Because MS-based proteomic analysis heavily depends on human protein databases available in public, new polymorphisms or mutations may not be listed in the databases and, thus, cannot be identified using the technique. In such situations, a separate workflow to compare the newly identified mutations against previously known variants is utilized.
