Contents



Estefania Azevedo, Priscila F. Silva, Fernando Palhano, Carolina A. Braga and Debora Foguel


Preface

deadly disease.

nosis and typing are made early.

care providers who take care of amyloid patients.

for her insightful suggestion, contribution and inspiration on the book.

Amyloidosis is an uncommon disorder characterized by the deposition of extracellular fibril amyloid proteins in various organs and tissues. It is estimated that there are about 3,000 new cases annually in the United States. Amyloidosis is classified by the precursor plasma proteins that form the extracellular fibril deposits. The primary systemic type, or the AL type, is due to monoclonal immunoglobulin free light chains. The hereditary type, also known as familial type, is mostly secondary to mutant transthyretin deposition while the wild type transthyretin type or also called senile type is due to normal wild-type transthyretin deposition. The secon‐ dary type or the AA type is related to amyloid A protein, which is an acute reactive protein. Dialysis related amyloidosis is caused by the deposition of β2-microglobulin, which has been increasingly recognized. Significant advancement has been made recently, which not only pro‐ vides insight into its pathophysiology but also helps to discover new therapies to fight the

Unfortunately, because of rarity of the disease and its protean clinical manifestations, many pa‐ tients were misdiagnosed, especially at early stage of the disease. Consequently, many patients missed the opportunity window of effective therapy, in particularly the AL type. The aim of the book is to review the fascinating history and the mechanism of the disease; to help the readers become familiar with the clinical presentation; and to review the latest diagnostic and therapeu‐ tic development. Effective or even curative therapies are available, especially if accurate diag‐

I would like to thank all the authors who have contributed to the book and congratulate them on their excellent works. This volume provides a comprehensive update of our knowledge on amyloidosis of different types. It will be a useful daily reference for physician and other health

I would like to sincerely thank my parent and my family (Gloria, Amy, Emily and Eric) for their endless love and support. You spent more time waiting for me than being with me while I was studying, writing, and taking care of my patients. I would also like to thank my several men‐ tors, including Drs. Liu Lisheng, Zhu Guoying, Geoffrey Toffler, James Muller, Joseph Loscal‐ zo, Jae Oh, Rick Nishimura, Carole Warnes and Kyle Klarich. You continue to inspire me to become a better cardiologist and a better person. Finally, I greatly appreciate Dr. Cynthia Taub

**Dali Feng, MD, FACC.**

Minneapolis, Minnesota , USA

The Metropolitan Heart and Vascular Institute

Chapter 10 **Therapeutic Strategies in Amyloid A Amyloidosis Secondary to Rheumatoid Arthritis 213** Tadashi Nakamura

## Preface

Chapter 7 **Anti-Cytokine Therapy for AA Amyloidosis 131**

Chapter 8 **Treatment of End Stage Heart Failure Related to Cardiac**

**Section 3 The Advancement of Therapy 153**

**Arthritis: State of the Art 187**

**Rheumatoid Arthritis 213**

Tadashi Nakamura

**Amyloidosis 155**

Kushwaha

Arimitsu

**VI** Contents

Keisuke Hagihara, Syota Kagawa, Yuki Kishida and Junsuke

Tal Hasin, Eugenia Raichlin, Angela Dispenzieri and Sudhir

Chapter 9 **Diagnosis and Treatment of AA Amyloidosis with Rheumatoid**

Chapter 10 **Therapeutic Strategies in Amyloid A Amyloidosis Secondary to**

Takeshi Kuroda, Yoko Wada and Masaaki Nakano

Amyloidosis is an uncommon disorder characterized by the deposition of extracellular fibril amyloid proteins in various organs and tissues. It is estimated that there are about 3,000 new cases annually in the United States. Amyloidosis is classified by the precursor plasma proteins that form the extracellular fibril deposits. The primary systemic type, or the AL type, is due to monoclonal immunoglobulin free light chains. The hereditary type, also known as familial type, is mostly secondary to mutant transthyretin deposition while the wild type transthyretin type or also called senile type is due to normal wild-type transthyretin deposition. The secon‐ dary type or the AA type is related to amyloid A protein, which is an acute reactive protein. Dialysis related amyloidosis is caused by the deposition of β2-microglobulin, which has been increasingly recognized. Significant advancement has been made recently, which not only pro‐ vides insight into its pathophysiology but also helps to discover new therapies to fight the deadly disease.

Unfortunately, because of rarity of the disease and its protean clinical manifestations, many pa‐ tients were misdiagnosed, especially at early stage of the disease. Consequently, many patients missed the opportunity window of effective therapy, in particularly the AL type. The aim of the book is to review the fascinating history and the mechanism of the disease; to help the readers become familiar with the clinical presentation; and to review the latest diagnostic and therapeu‐ tic development. Effective or even curative therapies are available, especially if accurate diag‐ nosis and typing are made early.

I would like to thank all the authors who have contributed to the book and congratulate them on their excellent works. This volume provides a comprehensive update of our knowledge on amyloidosis of different types. It will be a useful daily reference for physician and other health care providers who take care of amyloid patients.

I would like to sincerely thank my parent and my family (Gloria, Amy, Emily and Eric) for their endless love and support. You spent more time waiting for me than being with me while I was studying, writing, and taking care of my patients. I would also like to thank my several men‐ tors, including Drs. Liu Lisheng, Zhu Guoying, Geoffrey Toffler, James Muller, Joseph Loscal‐ zo, Jae Oh, Rick Nishimura, Carole Warnes and Kyle Klarich. You continue to inspire me to become a better cardiologist and a better person. Finally, I greatly appreciate Dr. Cynthia Taub for her insightful suggestion, contribution and inspiration on the book.

> **Dali Feng, MD, FACC.** The Metropolitan Heart and Vascular Institute Minneapolis, Minnesota , USA

**Section 1**

**History and Clinical Diagnosis and Typing**

**History and Clinical Diagnosis and Typing**

**Chapter 1**

**"Amyloid" — Historical Aspects**

Additional information is available at the end of the chapter

General agreement prevails today on the contents of the term "amyloid". It refers to "a condition associated with a number of inherited and inflammatory disorders in which extracellular deposits of fibrillar proteins are responsible for tissue damage and functional compromise", as defined in the textbook of pathology [1]. One and half centuries ago, in contrast, the nature of amyloid was the very target of an academic dispute among the leading scientists, European at those days. Curiously, the term "amyloid" has prevailed although in

The term "amyloid" was brought in the scientific literature by the German botanist Matthias Schleiden (1804 - 1881). Schleiden was born in Hamburg as the son of a Hamburger physician. He first studied laws in Heidelberg and received his pHD in 1826. However, working as a lawyer felt unsatisfactory to him and he turned to study medicine in 1832, in Göttingen and Berlin. Schleiden oriented to botany, microscopy and anatomy, with a special interest in the chemical and anatomical composition of plant cell, and received his second PhD in 1839. One of Schleiden's major ideas was to apply the iodine-sulphuric acid test for starch in plants. This test was originally described in 1814 by Colin and Gaultier de Claubry to show the blue staining reaction of starch with iodine and sulphuric acid [2]. Schleiden presented his discoveries at the scientific meetings of the "Gesellschaft Naturforschender Freunde" and reported on the application of the iodine-sulphuric acid test on plants on the 20th February, 1838 (in: Ostwalds klassiker der exakten Wissenschaften, band 275 Verlag Harri Deutsch (Klassische Schriften zur zellenlehre. Matthias Jacob Scleiden, Theodor Schwann, Max Schulze, text in German), cited in [3]. Scleiden's original interpretation was that the reaction demonstrated the transformation

> © 2013 Tanskanen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

the course of time the concept of amyloid has nearly turned upside down.

**2. Origin of amyloid: Matthias Schleiden and botany**

Maarit Tanskanen

**1. Introduction**

http://dx.doi.org/10.5772/53423

## **Chapter 1**

## **"Amyloid" — Historical Aspects**

## Maarit Tanskanen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53423

## **1. Introduction**

General agreement prevails today on the contents of the term "amyloid". It refers to "a condition associated with a number of inherited and inflammatory disorders in which extracellular deposits of fibrillar proteins are responsible for tissue damage and functional compromise", as defined in the textbook of pathology [1]. One and half centuries ago, in contrast, the nature of amyloid was the very target of an academic dispute among the leading scientists, European at those days. Curiously, the term "amyloid" has prevailed although in the course of time the concept of amyloid has nearly turned upside down.

## **2. Origin of amyloid: Matthias Schleiden and botany**

The term "amyloid" was brought in the scientific literature by the German botanist Matthias Schleiden (1804 - 1881). Schleiden was born in Hamburg as the son of a Hamburger physician. He first studied laws in Heidelberg and received his pHD in 1826. However, working as a lawyer felt unsatisfactory to him and he turned to study medicine in 1832, in Göttingen and Berlin. Schleiden oriented to botany, microscopy and anatomy, with a special interest in the chemical and anatomical composition of plant cell, and received his second PhD in 1839. One of Schleiden's major ideas was to apply the iodine-sulphuric acid test for starch in plants. This test was originally described in 1814 by Colin and Gaultier de Claubry to show the blue staining reaction of starch with iodine and sulphuric acid [2]. Schleiden presented his discoveries at the scientific meetings of the "Gesellschaft Naturforschender Freunde" and reported on the application of the iodine-sulphuric acid test on plants on the 20th February, 1838 (in: Ostwalds klassiker der exakten Wissenschaften, band 275 Verlag Harri Deutsch (Klassische Schriften zur zellenlehre. Matthias Jacob Scleiden, Theodor Schwann, Max Schulze, text in German), cited in [3]. Scleiden's original interpretation was that the reaction demonstrated the transformation

© 2013 Tanskanen; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of the plant material into starch [4]. Schleiden published his several botanical findings in the book form in 1842-43, with the title "Grundzige der wissenschaftlichen Botanik". It is remark‐ able that the 2nd and 3rd editions of the book, subtitled as "Die Botanik als inductive Wissen‐ schaft behandelt" were also translated in English in 1849, with the name "Principles of Scientific Botany" and "Botany as an Inductive Science", reflecting the attraction that Schlei‐ den's observations woke also outside Germany. In the above mentioned book Schleiden first time uses the term "*amyloid*" for starch, referring to"starch-like". The word itself stems from the latin word "*amylym*" for starch. Schleiden describes "amyloid" to represent "a normal amylaceous constituent in plants" [2], as shown in the straight citing from the English translation of the book [5] below.

Virchow was interested in microscopic studies, similarly to Schleiden. Virchow used the word "amyloid" first time in 1854 in his publication "Über eine in Gehirn und Rückenmark des Menschen aufgefundene Substanz mit der chemischen Reaction der Cellulose", in Virchow's Archiv für Pathologische Anatomie and Physiologie und fur klinische Medicin. Berlin 6; 354-368; 1854 [7]. In this paper Virchow described the small round deposits in the nervous system (Figure 1) with the mention that those structures showed the same color reaction with iodine and sulfuric acid, i.e. a change from brown to blue, typical to starch. Therefore, Virchow was convinced that those structures were identical to starch [8]. Virchow named those structures "corpora amylacea", similarly to Schleiden (the name based on the Latin term "amylum" for starch, see previously). Later Virchow applied the iodine sulphuric acid test to other tissues infiltrated with amyloid. The representatives of the French and British Schools instead considered amyloid to be more closely related to cellulose [2]. They use the name "lardaceous" (based on the bacon-like appearance of the tissue, French School) and "waxy" (based on the homogeneity of the material, British School). They could also use the term "sago"

"Amyloid" — Historical Aspects http://dx.doi.org/10.5772/53423 5

**Figure 1.** Corpora amylacea (arrows) stain blue in H&E (A) and brown in methenamine-silver (B) stain which also re‐ veals a few senile plaques of diffuse type (B). Diffuse plaques do not contain amyloid. The patient was 104-year old

female suffering from vascular dementia. Original magnification x 200.

(a sweet substance in certain palm species).

"Amyloid is, when dry, a cartilaginous, but moist, gelatinous, clear, transparent body, soluble in boiling water, strong acids, and caustic alkalies, but not in ether and alcohol in a concentrated state. It is coloured blue by iodine, and the combination is soluble in water, giving it a golden-yellow colour. It is found only in the layers of the primary cellmembrane. There is no chemical analysis of this substance. It has been found at present only in the cotyledon-cells of Schotia latifolia,S. speciosa, Hymencea Courbaril, Mucuna urens, M. gigantea, and Tamarindusindica."

The application of the iodine-sulphuric acid test on plants was not the most remarkable among Schleiden's scientific discoveries. Based on his interest in microscopic studies he got the unique idea that plants are made of cells, and that the growth of plants depends on the production of new cells. To get to this idea Schleiden was also lucky. In Berlin he had met Theodor Schwann (1810 - 1882), another great scientist of those days who had made similar observations in animals [6]. The published observations of Schleiden (1838) and Schwann (1839) form the basis for the unified "cell theory", applicable to all living organisms. During the same time (1839) the French chemist Anselme Payen (1795 - 1878) described a substance in woods that resembled starch. This substance reacted with iodine-sulphuric acid test similarly to starch, and Payen named it "cellulose". The iodine-sulphuric acid reaction became later on a standard procedure used by botanists to demonstrate the presence of cellulose in woods [4].

It is well possible that amyloid deposits have been described even earlier, in the reports on human autopsy cases with homogenous material in liver or spleen tissue [2], probably representing amyloid. The first observation stems from the year 1639, described by Nicolaus Fontanus.

## **3. The term "amyloid" in the medical literature: Rudolf Virchow**

Whereas Matthias Schleiden was the first to use the term "amyloid" in botanics, it was the German pathologist Rudolf Virchow (1804 - 1881) who applied it in the medical literature. Virchow studied medicine and anatomy in Berlin and Würzburg, and was graduated in 1843. Virchow was interested in microscopic studies, similarly to Schleiden. Virchow used the word "amyloid" first time in 1854 in his publication "Über eine in Gehirn und Rückenmark des Menschen aufgefundene Substanz mit der chemischen Reaction der Cellulose", in Virchow's Archiv für Pathologische Anatomie and Physiologie und fur klinische Medicin. Berlin 6; 354-368; 1854 [7]. In this paper Virchow described the small round deposits in the nervous system (Figure 1) with the mention that those structures showed the same color reaction with iodine and sulfuric acid, i.e. a change from brown to blue, typical to starch. Therefore, Virchow was convinced that those structures were identical to starch [8]. Virchow named those structures "corpora amylacea", similarly to Schleiden (the name based on the Latin term "amylum" for starch, see previously). Later Virchow applied the iodine sulphuric acid test to other tissues infiltrated with amyloid. The representatives of the French and British Schools instead considered amyloid to be more closely related to cellulose [2]. They use the name "lardaceous" (based on the bacon-like appearance of the tissue, French School) and "waxy" (based on the homogeneity of the material, British School). They could also use the term "sago" (a sweet substance in certain palm species).

of the plant material into starch [4]. Schleiden published his several botanical findings in the book form in 1842-43, with the title "Grundzige der wissenschaftlichen Botanik". It is remark‐ able that the 2nd and 3rd editions of the book, subtitled as "Die Botanik als inductive Wissen‐ schaft behandelt" were also translated in English in 1849, with the name "Principles of Scientific Botany" and "Botany as an Inductive Science", reflecting the attraction that Schlei‐ den's observations woke also outside Germany. In the above mentioned book Schleiden first time uses the term "*amyloid*" for starch, referring to"starch-like". The word itself stems from the latin word "*amylym*" for starch. Schleiden describes "amyloid" to represent "a normal amylaceous constituent in plants" [2], as shown in the straight citing from the English

"Amyloid is, when dry, a cartilaginous, but moist, gelatinous, clear, transparent body, soluble in boiling water, strong

acids, and caustic alkalies, but not in ether and alcohol in a concentrated state. It is coloured blue by iodine, and the

combination is soluble in water, giving it a golden-yellow colour. It is found only in the layers of the primary cell-

membrane. There is no chemical analysis of this substance. It has been found at present only in the cotyledon-cells of

The application of the iodine-sulphuric acid test on plants was not the most remarkable among Schleiden's scientific discoveries. Based on his interest in microscopic studies he got the unique idea that plants are made of cells, and that the growth of plants depends on the production of new cells. To get to this idea Schleiden was also lucky. In Berlin he had met Theodor Schwann (1810 - 1882), another great scientist of those days who had made similar observations in animals [6]. The published observations of Schleiden (1838) and Schwann (1839) form the basis for the unified "cell theory", applicable to all living organisms. During the same time (1839) the French chemist Anselme Payen (1795 - 1878) described a substance in woods that resembled starch. This substance reacted with iodine-sulphuric acid test similarly to starch, and Payen named it "cellulose". The iodine-sulphuric acid reaction became later on a standard procedure

It is well possible that amyloid deposits have been described even earlier, in the reports on human autopsy cases with homogenous material in liver or spleen tissue [2], probably representing amyloid. The first observation stems from the year 1639, described by Nicolaus

Whereas Matthias Schleiden was the first to use the term "amyloid" in botanics, it was the German pathologist Rudolf Virchow (1804 - 1881) who applied it in the medical literature. Virchow studied medicine and anatomy in Berlin and Würzburg, and was graduated in 1843.

Schotia latifolia,S. speciosa, Hymencea Courbaril, Mucuna urens, M. gigantea, and Tamarindusindica."

used by botanists to demonstrate the presence of cellulose in woods [4].

**3. The term "amyloid" in the medical literature: Rudolf Virchow**

translation of the book [5] below.

4 Amyloidosis

Fontanus.

**Figure 1.** Corpora amylacea (arrows) stain blue in H&E (A) and brown in methenamine-silver (B) stain which also re‐ veals a few senile plaques of diffuse type (B). Diffuse plaques do not contain amyloid. The patient was 104-year old female suffering from vascular dementia. Original magnification x 200.

Virchow developed an observational and experimental view on medical sciences. In this regard he resembled the French and British scientists at that time but contrasted to the more speculative German scientific tradition. As Virchow's writings received an unfavorable attention in German journals he decided to found in 1847 a journal of his own "*Archiv für pathologische Anatomie und Physiologie und für klinische Medizin"* with another German pathol‐ ogist, Benno Reinhardt. The bearing idea of the new journal was not to publish papers containing "outdated, untested, dogmatic or speculative ideas". After Reinhardt's death in 1852 Virchow edited the paper alone, with the name "Virchow's Archives", a world-famous and respected journal still today.


"Amyloid" — Historical Aspects http://dx.doi.org/10.5772/53423 7

There was another novel invention that Virchow did not accept: the metachromatic stains. In 1875 three scientists; the French pathologist and histologist Victor Cornil (1837 - 1908) in Paris, the Austrian anatomist Richard Heschl (1824 - 1881) in Vienna and Rudolf Jürgens in Berlin described independently the usefulness of methylviolet stain to detect amyloid. Already next year, in 1876, Soyka reported having found amyloid in the cardiac tissue with the use of this new method (Soyka J. Prag Med Wschr 1: 165, 1876; cited in Hodgkinson and Buerger [17,18]). William Ackroyd and Paul Ehrlich described methylviolet stain as "metachromatic" in 1878. Metachromatic stains challenged Virchow's iodine sulfuric acid test for decades [2] but were

**5. Development of contemporary staining methods: Congo red dye and**

Reactivity with Congo red stain or "Congophilia with apple green birefringence" was the first criterion for amyloid [11], introduced by the Belgian Physician Paul Divry (Divry P. Etude histo-chimique des plaques seniles. J de Neurologie et de Psychiatrie 27:643-57, 1927, cited in Sipe [11]). Congo red dye itself was invented by the German chemist Paul Böttiger in 1884 (Böttiger P. Deutsches Reich's Patent 28753, August 20, 1884, cited in Frid [19]). Congo red is an aniline dye, originally created and used for staining textiles. Böttiger developed the first "direct" dye that did not require additional substances for fixation to the textile fibers. The owner of the patent, the AGFA Corporation developed the name "Congo" to the new dye after the diplomatic conference that was ongoing in Berlin just at that time (1884 - 1885). The goal of the "Congo conference" was to mediate a trade dispute between several European colonial powers in the Congo River Basin in Central Africa [2,20]. The name "Congo" referred to an exotic place that was on the tip of the lips, and proved to be effective for marketing purposes [2,20]. In addition to staining textiles Congo red was actually used to stain tissues already in 1886 [20]. However, it was not until in the year 1922 when the young German chemist Herman Bennhold discovered the capacity of Congo red to bind to amyloid (Bennhold H. Eine spezifische Amyloidfärbung mit Kongorot. Münchener Medizinische Wochenschrift (Novem‐ ber):1537-1538, 1922; cited in Kyle [2]). In 1962 Puchtler described the renewed method for the

The Puchtler modification [21] of Congo red staining is widely used in pathology as the first step in detecting amyloid in histological specimens. Of course, individual laboratories may apply their own variant of the method. Congo red staining is also applicable to frozen sections and for staining devices. In the diagnostic purposes the formalin-fixed histological samples are generally embedded in paraffin, sectioned 5-8 μm thick slices, stained with Congo red, and viewed in a light microscope under polarized light in which amyloid can be seen as red to

a pale autopsy liver [2].

eventually replaced by Congo red.

**fluorescence microscopy**

use of Congo red in histological preparations [21].

green birefringent homogeneous material.

Virchow's investigations in pathology extended to several other clinically significant issues. For instance, he discovered the mechanism of thromboembolism and developed the standard method of autopsy, as described in "The handbook on special pathology and therapeutics" in 1854. Further, in addition to Schleiden and Schwann, Virchow was the third scientist who has been nominated as an inventor of the "cell theory". He applied the concept in humans and published it in "Cellular pathology" in 1859. Yet, the probably most significant of Virchow's ideas was that he understood to combine the macroscopic and microscopic pathologies with clinical manifestations of disease [9].

Virchow was not only a pathologist. His interest and knowledge extended to anthropology, archeology, politics and social sciences [10]. For instance, Virchow established the first hospital trains bringing medicine in the battlefields, and he also was the first to understand the influence of poor hygiene on the spread of contagious illnesses. Political and social activities combined with huge scientific career brought to Virchow the status as the world-renowned physician and "Father of pathology".

## **4. The end of the 1800's: Progress in the staining methods to detect amyloid**

A new insight into the biochemical character of amyloid was presented in 1859 when the prominent German chemist August Kekulé (1829 - 1896) reported on the high proportion of nitrogen in organs infiltrated with amyloid [2,11]. Kekulé assumpted that the material mainly represented "albumoid" compounds. In addition, he did not find material corresponding chemically to "amylon" or cellulose." Virchow never agreed with Kekulé, criticizing his method to analyze the whole tissue specimens (e.g. liver). After Virchow's opinion, convincing results necessitated a method to isolate the amyloid substance first [2]. Indeed, also in this he was ahead of his time. After more than one hundred years other constituents such as glyco‐ saminoglycans and heparan sulphate [12,13] and chondroitin sulphate -containing proteogly‐ cans [14,15] were identified as additional, albeit minor components of amyloid deposits [16].

In contrast to Virchow, Kekulé's observation on high nitrogen contents of amyloid were accepted by several scientists, including the British physician Samuel Wilks (1824 - 1919) who had collected more than 60 cases with the white, "stony", "gelatinous" or "lardaceous" visceral material, i.e. amyloid detected at the autopsy [2]. George Budd, another British internist (1808 - 1882) actually got the same result than Kekulé when analyzing the chemical composition of a pale autopsy liver [2].

Virchow developed an observational and experimental view on medical sciences. In this regard he resembled the French and British scientists at that time but contrasted to the more speculative German scientific tradition. As Virchow's writings received an unfavorable attention in German journals he decided to found in 1847 a journal of his own "*Archiv für pathologische Anatomie und Physiologie und für klinische Medizin"* with another German pathol‐ ogist, Benno Reinhardt. The bearing idea of the new journal was not to publish papers containing "outdated, untested, dogmatic or speculative ideas". After Reinhardt's death in 1852 Virchow edited the paper alone, with the name "Virchow's Archives", a world-famous

Virchow's investigations in pathology extended to several other clinically significant issues. For instance, he discovered the mechanism of thromboembolism and developed the standard method of autopsy, as described in "The handbook on special pathology and therapeutics" in 1854. Further, in addition to Schleiden and Schwann, Virchow was the third scientist who has been nominated as an inventor of the "cell theory". He applied the concept in humans and published it in "Cellular pathology" in 1859. Yet, the probably most significant of Virchow's ideas was that he understood to combine the macroscopic and microscopic pathologies with

Virchow was not only a pathologist. His interest and knowledge extended to anthropology, archeology, politics and social sciences [10]. For instance, Virchow established the first hospital trains bringing medicine in the battlefields, and he also was the first to understand the influence of poor hygiene on the spread of contagious illnesses. Political and social activities combined with huge scientific career brought to Virchow the status as the world-renowned physician

**4. The end of the 1800's: Progress in the staining methods to detect amyloid**

A new insight into the biochemical character of amyloid was presented in 1859 when the prominent German chemist August Kekulé (1829 - 1896) reported on the high proportion of nitrogen in organs infiltrated with amyloid [2,11]. Kekulé assumpted that the material mainly represented "albumoid" compounds. In addition, he did not find material corresponding chemically to "amylon" or cellulose." Virchow never agreed with Kekulé, criticizing his method to analyze the whole tissue specimens (e.g. liver). After Virchow's opinion, convincing results necessitated a method to isolate the amyloid substance first [2]. Indeed, also in this he was ahead of his time. After more than one hundred years other constituents such as glyco‐ saminoglycans and heparan sulphate [12,13] and chondroitin sulphate -containing proteogly‐ cans [14,15] were identified as additional, albeit minor components of amyloid deposits [16].

In contrast to Virchow, Kekulé's observation on high nitrogen contents of amyloid were accepted by several scientists, including the British physician Samuel Wilks (1824 - 1919) who had collected more than 60 cases with the white, "stony", "gelatinous" or "lardaceous" visceral material, i.e. amyloid detected at the autopsy [2]. George Budd, another British internist (1808

and respected journal still today.

6 Amyloidosis

clinical manifestations of disease [9].

and "Father of pathology".

There was another novel invention that Virchow did not accept: the metachromatic stains. In 1875 three scientists; the French pathologist and histologist Victor Cornil (1837 - 1908) in Paris, the Austrian anatomist Richard Heschl (1824 - 1881) in Vienna and Rudolf Jürgens in Berlin described independently the usefulness of methylviolet stain to detect amyloid. Already next year, in 1876, Soyka reported having found amyloid in the cardiac tissue with the use of this new method (Soyka J. Prag Med Wschr 1: 165, 1876; cited in Hodgkinson and Buerger [17,18]). William Ackroyd and Paul Ehrlich described methylviolet stain as "metachromatic" in 1878. Metachromatic stains challenged Virchow's iodine sulfuric acid test for decades [2] but were eventually replaced by Congo red.

## **5. Development of contemporary staining methods: Congo red dye and fluorescence microscopy**

Reactivity with Congo red stain or "Congophilia with apple green birefringence" was the first criterion for amyloid [11], introduced by the Belgian Physician Paul Divry (Divry P. Etude histo-chimique des plaques seniles. J de Neurologie et de Psychiatrie 27:643-57, 1927, cited in Sipe [11]). Congo red dye itself was invented by the German chemist Paul Böttiger in 1884 (Böttiger P. Deutsches Reich's Patent 28753, August 20, 1884, cited in Frid [19]). Congo red is an aniline dye, originally created and used for staining textiles. Böttiger developed the first "direct" dye that did not require additional substances for fixation to the textile fibers. The owner of the patent, the AGFA Corporation developed the name "Congo" to the new dye after the diplomatic conference that was ongoing in Berlin just at that time (1884 - 1885). The goal of the "Congo conference" was to mediate a trade dispute between several European colonial powers in the Congo River Basin in Central Africa [2,20]. The name "Congo" referred to an exotic place that was on the tip of the lips, and proved to be effective for marketing purposes [2,20]. In addition to staining textiles Congo red was actually used to stain tissues already in 1886 [20]. However, it was not until in the year 1922 when the young German chemist Herman Bennhold discovered the capacity of Congo red to bind to amyloid (Bennhold H. Eine spezifische Amyloidfärbung mit Kongorot. Münchener Medizinische Wochenschrift (Novem‐ ber):1537-1538, 1922; cited in Kyle [2]). In 1962 Puchtler described the renewed method for the use of Congo red in histological preparations [21].

The Puchtler modification [21] of Congo red staining is widely used in pathology as the first step in detecting amyloid in histological specimens. Of course, individual laboratories may apply their own variant of the method. Congo red staining is also applicable to frozen sections and for staining devices. In the diagnostic purposes the formalin-fixed histological samples are generally embedded in paraffin, sectioned 5-8 μm thick slices, stained with Congo red, and viewed in a light microscope under polarized light in which amyloid can be seen as red to green birefringent homogeneous material.

(Figure 2). Interestingly, the light microscope finding has been observed to vary in different types of transthyretin (TTR)-related amyloidosis and accordingly the distribution into two different histological patterns of amyloid deposition (designed as A and B) has been proposed [22]. In pattern A, seen in senile systemic amyloidosis (SSA) and in some cases with TTR-related familial amyloidosis, there is weakly congophilic, homogenous amyloid material that is patchy distributed. In pattern B, detected in a part of patients with TTR-related familial disease, strongly congophilic amyloid appears as thin streaks. Thus, the biochemical structure of amyloid fibrils can be transmitted to the microscopic finding.

as well. The binding of Congo red to amyloid induces a characteristic shift in the maximal optical absorbance of the molecule from 490 nm to 540 nm. The mechanisms of interaction between Congo red and amyloid fibrils has been intensively studied [24,25] but the process is not completely understood [19]. Congo red binding has been assumed to depend on the secondary, β-pleated configuration of the fibril, possibly mediated by hydrophobic interac‐ tions of the benzidine centers as well as the electrostatically charged terminal groups [19].

"Amyloid" — Historical Aspects http://dx.doi.org/10.5772/53423 9

Amyloid can also be visualized using the fluorescence microscope. Fluorescence microscope is a light microscope used to study the properties of organic and inorganic substances with the aid of the phenomena of fluorescence and phosphorescence. The component of interest in the specimen is labeled with a fluorescent molecule, the "fluorophore". Amyloid can be detected using thioflavin stains (Thioflavin-T or -S) which emit green fluorescence when they are bound to amyloid. Thioflavin-T (Basic Yellow 1 or CI 49005) is a benzothiazole salt, obtained by methylating dehydrothiotoluidine with methanol in the presence of hydrochloric acid. When the dye binds to β sheets it undergoes a 120 nm red shift of its excitation spectrum that may selectively be excitated at 450 nm, resulting in a fluorescence signal at 482 nm. Thioflavin-S is a mixture of compounds resulting from the methylation of dehydrothiotoluidine with sulphonic acid. The fluorescence method is specific for amyloid similarly to Congo red [26] and very sensitive. The disappearance of fluorescence during time can be regarded a disad‐

vantage of the method, because the reaction cannot be re-examined later.

**pathologies**

amyloid formed on tau protein.

**6. The beginning of the 1900's: Alzheimer's disease and associated**

In 1907, Aloysius (Alois) Alzheimer described "senile" plaques and neurofibrillary tangles in a demented patient. Today we know that the plaques represent *extracellular* amyloid derived from amyloid beta (Aβ) protein whereas the neurofibrillary tangles represent *intracellular*

Alzheimer (1864 -1915) was German psychiatrist, born in Bavaria. He got his medical education at the universities of Tübingen and also in Berlin and Würzburg, similarly to Virchow, to receive his medical degree in 1887. Soon thereafter he began to work in a mental asylum "die Städtische Anstalt für Irre und Epileptische" in Frankfurt am Main. Alzheimer's scientific interest focused on pathology of the nervous system, especially anatomy of the cerebral cortex. He collaborated with the neuropathologist Franz Nissl (1860 - 1919) and learned Nissl's method of silver staining of the histological sections. In the year 1901 Alzheimer happened to get the 51 -year old Mrs. Auguste Deter to be his patient at the Frankfurt Asylum. Mrs. Deter had a very unusual clinical picture with loss of short-term memory and odd behavioral symptoms. In 1902 Alzheimer moved to work with his colleague, another German psychiatrist Emil Kraepelin (1856 - 1926) at the University of Heidelberg. Kraepelin had, similarly to Alzheimer, special interest in neuropathology. Both moved to Munich next year. Mrs Deter died in 1906 in Frankfurt, and Alzheimer decided to bring her brain and medical records to Munich for neuropathological study. He grasped to apply Nissl's method of silver staining on the

**Figure 2.** The red colour (A) of amyloid in the cardiac tissue of a patient with senile systemic amyloidosis (SSA) gradu‐ ally (B,C) turns to green (D) in the polarized light. Original magnification x 400.

The chemical name of Congo red, also known as "direct red", "direct red 28", or "cotton red", is 3,3΄-[(1,1´-biphenyl)-4,4´-diylbis(azo)] *bis*-(4-amino-1-naphtalene acid) disodium salt (C32H22N6O6S2 2Na). It is a symmetrical molecule with the molecular weight of 696.7 g/mol and the diameter approximately 21Å [23]. The molecule has a hydrophobic center composed of two phenyl rings that are linked via diazo bonds to two charged terminal naphtalene moieties. The terminal parts of Congo red contain sulphonic acid and amine groups. Congo red exists in chinone form in acidic solution, and in sulphonazo form in basic solution, changing the color from blue (below pH 3) to red (above pH 5). Thus, Congo red can be used as a pH indicator as well. The binding of Congo red to amyloid induces a characteristic shift in the maximal optical absorbance of the molecule from 490 nm to 540 nm. The mechanisms of interaction between Congo red and amyloid fibrils has been intensively studied [24,25] but the process is not completely understood [19]. Congo red binding has been assumed to depend on the secondary, β-pleated configuration of the fibril, possibly mediated by hydrophobic interac‐ tions of the benzidine centers as well as the electrostatically charged terminal groups [19].

(Figure 2). Interestingly, the light microscope finding has been observed to vary in different types of transthyretin (TTR)-related amyloidosis and accordingly the distribution into two different histological patterns of amyloid deposition (designed as A and B) has been proposed [22]. In pattern A, seen in senile systemic amyloidosis (SSA) and in some cases with TTR-related familial amyloidosis, there is weakly congophilic, homogenous amyloid material that is patchy distributed. In pattern B, detected in a part of patients with TTR-related familial disease, strongly congophilic amyloid appears as thin streaks. Thus, the biochemical structure of

**Figure 2.** The red colour (A) of amyloid in the cardiac tissue of a patient with senile systemic amyloidosis (SSA) gradu‐

The chemical name of Congo red, also known as "direct red", "direct red 28", or "cotton red", is 3,3΄-[(1,1´-biphenyl)-4,4´-diylbis(azo)] *bis*-(4-amino-1-naphtalene acid) disodium salt (C32H22N6O6S2 2Na). It is a symmetrical molecule with the molecular weight of 696.7 g/mol and the diameter approximately 21Å [23]. The molecule has a hydrophobic center composed of two phenyl rings that are linked via diazo bonds to two charged terminal naphtalene moieties. The terminal parts of Congo red contain sulphonic acid and amine groups. Congo red exists in chinone form in acidic solution, and in sulphonazo form in basic solution, changing the color from blue (below pH 3) to red (above pH 5). Thus, Congo red can be used as a pH indicator

ally (B,C) turns to green (D) in the polarized light. Original magnification x 400.

amyloid fibrils can be transmitted to the microscopic finding.

8 Amyloidosis

Amyloid can also be visualized using the fluorescence microscope. Fluorescence microscope is a light microscope used to study the properties of organic and inorganic substances with the aid of the phenomena of fluorescence and phosphorescence. The component of interest in the specimen is labeled with a fluorescent molecule, the "fluorophore". Amyloid can be detected using thioflavin stains (Thioflavin-T or -S) which emit green fluorescence when they are bound to amyloid. Thioflavin-T (Basic Yellow 1 or CI 49005) is a benzothiazole salt, obtained by methylating dehydrothiotoluidine with methanol in the presence of hydrochloric acid. When the dye binds to β sheets it undergoes a 120 nm red shift of its excitation spectrum that may selectively be excitated at 450 nm, resulting in a fluorescence signal at 482 nm. Thioflavin-S is a mixture of compounds resulting from the methylation of dehydrothiotoluidine with sulphonic acid. The fluorescence method is specific for amyloid similarly to Congo red [26] and very sensitive. The disappearance of fluorescence during time can be regarded a disad‐ vantage of the method, because the reaction cannot be re-examined later.

## **6. The beginning of the 1900's: Alzheimer's disease and associated pathologies**

In 1907, Aloysius (Alois) Alzheimer described "senile" plaques and neurofibrillary tangles in a demented patient. Today we know that the plaques represent *extracellular* amyloid derived from amyloid beta (Aβ) protein whereas the neurofibrillary tangles represent *intracellular* amyloid formed on tau protein.

Alzheimer (1864 -1915) was German psychiatrist, born in Bavaria. He got his medical education at the universities of Tübingen and also in Berlin and Würzburg, similarly to Virchow, to receive his medical degree in 1887. Soon thereafter he began to work in a mental asylum "die Städtische Anstalt für Irre und Epileptische" in Frankfurt am Main. Alzheimer's scientific interest focused on pathology of the nervous system, especially anatomy of the cerebral cortex. He collaborated with the neuropathologist Franz Nissl (1860 - 1919) and learned Nissl's method of silver staining of the histological sections. In the year 1901 Alzheimer happened to get the 51 -year old Mrs. Auguste Deter to be his patient at the Frankfurt Asylum. Mrs. Deter had a very unusual clinical picture with loss of short-term memory and odd behavioral symptoms. In 1902 Alzheimer moved to work with his colleague, another German psychiatrist Emil Kraepelin (1856 - 1926) at the University of Heidelberg. Kraepelin had, similarly to Alzheimer, special interest in neuropathology. Both moved to Munich next year. Mrs Deter died in 1906 in Frankfurt, and Alzheimer decided to bring her brain and medical records to Munich for neuropathological study. He grasped to apply Nissl's method of silver staining on the histological sections of Mrs. Deter's brains, and thereby identified the neurofibrillary deposits in the atrophic brain. The first report of the extraordinary pathological findings was presented in the same year at the University of Tübingen, prior to the appearance of the publication in 1907 (Alzheimer A. Über eine eigenartige Erkrankung der Hirnrinde. Allgemeine Zeitschrift für Psychtiatrie und Psychisch-gerichtliche Medizin. 1907 Jan; 64:146-8).

The original histological sections on which Alzheimer based his description were rediscovered in the 1990ies in Munich. This gave the unique opportunity to re-evaluate his work [27]. Silver stains have been used to diagnose Alzheimer's disease (AD) during decades and the original observations made by Alzheimer's and Kraepelin are valid even today. Quite recently, techniques using the immunohistochemistry (IHC) -based techniques in the diagnosis of AD pathology have also been introduced [28]. Alzheimer's neuropathological discoveries were not restricted to AD pathology. For example, he also described the loss of nerve cells in the corpus striatum in Huntington's disease and brain changes in epilepsy [29].

Mrs. Deter suffered from a syndrome that is called today as *presenile* dementia. Presenile dementias form a group of hereditary dementia syndromes which are often autosomally dominantly inherited. It has turned out that presenile dementia syndrome is quite common in the population called Volga Germans (VG). VG stems from people who emigrated in the 1760 s from the German Hesse area around Frankfurt to the southern Volga region in Russia. During the late 19th and early 20th centuries many of the descendants of VG emigrated to US. Presenile dementia of the VG is due to the mutation N141I in the gene for presenilin (PSEN) 2 [30]. Interestingly, neuropathology of the brains of subjects with this mutation is similar to Mrs. Deter's case [27]. Therefore, especially as Mrs. Deter was living in the Hesse area in Germany, an idea was got that she would have belonged to the population of VG. Mrs. Deter's brain tissue was tested for the presence of the PSEN 2 mutation – but the result was negative [31]. Yet, this does not preclude that Mrs. Deter would have had a different mutation in PSEN 2 or a mutation in other genes such as PSEN 1 or APP.

**Figure 3.** A senile plaque with amyloid core stained with methenamine-silver (A), immunohistochemistry against Aβ

"Amyloid" — Historical Aspects http://dx.doi.org/10.5772/53423 11

Afterthe2ndworldwaramyloidresearchstretchedfromEuropetoincludealsoNorthernAmerica and Japan. A substantial advance in the field took place in the late fifties. Two American researchers, Alan S Cohen from Harvard Medical School in Boston and Evan Calkins from Massachusetts General Hospital reported on the fibrillary structures in the samples of several typesofamyloidsin1959usingelectronmicroscopy(CohenASandCalcinsE.Electronmicroscop‐ ic observations on a fibrous component in amyloid of diverse origins. Nature 183 1202-3, 1959; cited in Vinters [38]). Several attempts followed to isolate the fibrils from tissues and organs. Cohen and Calkins themselves described the first extraction method. It consisted of gentle physical separation and homogenization of the material in saline, followed by low-speed centrifugation. This yielded a layer of fibrils not present in the sedimentation pellets of normal tissues and demonstrated a green birefringence in polarized light after staining with Congo red [39]. The next method was published by George Glenner and Howard Bladen (NIA, Bethesda, Maryland). They had extracted amyloid fibrils using alkaline sodium glycinate in 1966 [40].

(B) and Congo red (C without and D with polarization) stains. Original magnification x 600.

**7. Identification and extraction of the amyloid fibril**

Although the diagnosis of Mrs. Deter is still open, Alzheimer's paper was a starting point to enormous amounts of experimental and applied investigations of AD, an old age associated dementia syndrome. The disease belongs to the major causes of death in the western world, and it is estimated that about 24 million people suffer from it worldwide [32]. In spite of the huge work, the etiology of AD is still uncertain. Several hypotheses have been proposed [33], of which the "amyloid cascade hypothesis" by John Hardy and Gerald Higgins [34] has maybe got the greatest attention. The theory has recently also been criticized [35] as many therapeutic attempts based on it have failed [36].

The first descriptions of AD pathology were based on silver staining (Figure 3A) without no idea about the biochemical composition (Figure 3B) or relationship to amyloid of such structures (Figure 3C,D). Plaque amyloid however was discovered relatively soon (in 1927, see previously) by Divry after Bennhod had published his application of Congo red in tissue (in 1922, see previously). Cerebrovascular amyloid (cerebral amyloid angiopathy, CAA), detect‐ able in 80-90% in the brains of patients with AD was first time reported by Greek Pantelakis in 1954 [37].

**Figure 3.** A senile plaque with amyloid core stained with methenamine-silver (A), immunohistochemistry against Aβ (B) and Congo red (C without and D with polarization) stains. Original magnification x 600.

## **7. Identification and extraction of the amyloid fibril**

histological sections of Mrs. Deter's brains, and thereby identified the neurofibrillary deposits in the atrophic brain. The first report of the extraordinary pathological findings was presented in the same year at the University of Tübingen, prior to the appearance of the publication in 1907 (Alzheimer A. Über eine eigenartige Erkrankung der Hirnrinde. Allgemeine Zeitschrift

The original histological sections on which Alzheimer based his description were rediscovered in the 1990ies in Munich. This gave the unique opportunity to re-evaluate his work [27]. Silver stains have been used to diagnose Alzheimer's disease (AD) during decades and the original observations made by Alzheimer's and Kraepelin are valid even today. Quite recently, techniques using the immunohistochemistry (IHC) -based techniques in the diagnosis of AD pathology have also been introduced [28]. Alzheimer's neuropathological discoveries were not restricted to AD pathology. For example, he also described the loss of nerve cells in the

Mrs. Deter suffered from a syndrome that is called today as *presenile* dementia. Presenile dementias form a group of hereditary dementia syndromes which are often autosomally dominantly inherited. It has turned out that presenile dementia syndrome is quite common in the population called Volga Germans (VG). VG stems from people who emigrated in the 1760 s from the German Hesse area around Frankfurt to the southern Volga region in Russia. During the late 19th and early 20th centuries many of the descendants of VG emigrated to US. Presenile dementia of the VG is due to the mutation N141I in the gene for presenilin (PSEN) 2 [30]. Interestingly, neuropathology of the brains of subjects with this mutation is similar to Mrs. Deter's case [27]. Therefore, especially as Mrs. Deter was living in the Hesse area in Germany, an idea was got that she would have belonged to the population of VG. Mrs. Deter's brain tissue was tested for the presence of the PSEN 2 mutation – but the result was negative [31]. Yet, this does not preclude that Mrs. Deter would have had a different mutation in PSEN 2 or

Although the diagnosis of Mrs. Deter is still open, Alzheimer's paper was a starting point to enormous amounts of experimental and applied investigations of AD, an old age associated dementia syndrome. The disease belongs to the major causes of death in the western world, and it is estimated that about 24 million people suffer from it worldwide [32]. In spite of the huge work, the etiology of AD is still uncertain. Several hypotheses have been proposed [33], of which the "amyloid cascade hypothesis" by John Hardy and Gerald Higgins [34] has maybe got the greatest attention. The theory has recently also been criticized [35] as many therapeutic

The first descriptions of AD pathology were based on silver staining (Figure 3A) without no idea about the biochemical composition (Figure 3B) or relationship to amyloid of such structures (Figure 3C,D). Plaque amyloid however was discovered relatively soon (in 1927, see previously) by Divry after Bennhod had published his application of Congo red in tissue (in 1922, see previously). Cerebrovascular amyloid (cerebral amyloid angiopathy, CAA), detect‐ able in 80-90% in the brains of patients with AD was first time reported by Greek Pantelakis

für Psychtiatrie und Psychisch-gerichtliche Medizin. 1907 Jan; 64:146-8).

corpus striatum in Huntington's disease and brain changes in epilepsy [29].

a mutation in other genes such as PSEN 1 or APP.

attempts based on it have failed [36].

in 1954 [37].

10 Amyloidosis

Afterthe2ndworldwaramyloidresearchstretchedfromEuropetoincludealsoNorthernAmerica and Japan. A substantial advance in the field took place in the late fifties. Two American researchers, Alan S Cohen from Harvard Medical School in Boston and Evan Calkins from Massachusetts General Hospital reported on the fibrillary structures in the samples of several typesofamyloidsin1959usingelectronmicroscopy(CohenASandCalcinsE.Electronmicroscop‐ ic observations on a fibrous component in amyloid of diverse origins. Nature 183 1202-3, 1959; cited in Vinters [38]). Several attempts followed to isolate the fibrils from tissues and organs. Cohen and Calkins themselves described the first extraction method. It consisted of gentle physical separation and homogenization of the material in saline, followed by low-speed centrifugation. This yielded a layer of fibrils not present in the sedimentation pellets of normal tissues and demonstrated a green birefringence in polarized light after staining with Congo red [39]. The next method was published by George Glenner and Howard Bladen (NIA, Bethesda, Maryland). They had extracted amyloid fibrils using alkaline sodium glycinate in 1966 [40].

A significant step forward took place when M Pras (originally from the Tel Hashomer Hospital, Tel Aviv, Israel) and colleagues from New York University School of Medicine described the method to extract proteins from amyloid-laden tissues using water [41]. Spleen tissue from a deceased patient with "primary" (i.e. AA) amyloidosis was homogenized with physiological saline (NaCl), and the mixture was centrifuged. The sediment was next homogenized with NaCl several times to remove most of the soluble proteins and other soluble materials. Salt was then removed by homogenizing the residue in distilled water, followed by centrifugation of the suspension. Lastly, the residue was homogenized in distilled water and centrifuged four times to give a supernatant rich in protein. Adding Congo red dye and NaCl then resulted in a gelatinous precipitate with the typical green birefringence, demonstrating that the superna‐ tant represented soluble amyloid.

amyloid polyneuropathy (FAP) [52], the clinical condition having been described already in 1951 (Corino de Andrade, M. Preliminary note on an unusual form of peripheral neuropathy. Rev Neurol (Paris) 85: 302-6, 1951; cited in Kyle [2]). Similar diseases were found especially in Japan and Sweden in the subsequent decades. The Finnish type of familial amyloidosis, today known as AGel amyloidosis, was described in 1969 by the Finnish ophthalmologist Jouko Meretoja [53]. In 1980, TTR was characterized as the amyloid protein also in "senile cardiac amyloidosis" (SCA) [54], later renamed as senile systemic amyloidosis (SSA) [55,56]. In 1983, the Icelandic type of familial cerebral amyloid angiopathy (HCHWA-I) was found to be related to cystatin-C protein [57]. Next year, 1984, the first report on the AD-associated Aβ protein was published two Glenner and Wong who identified Aβ in the cerebrovascular tissue [58]. Colin Masters (Australian), Konrad Beyreuther (German) and colleagues described the same protein one year later, in 1985, in the plaques (Figure 3B). Japanese Fumitage Gejyo described beta 2 microglobulin as the amyloid fibril protein in the dialysis-related amyloid arthropathy in the same year [60]. Tau protein of the neurofibrillary tangles was identified in 1986 in the laborato‐

"Amyloid" — Historical Aspects http://dx.doi.org/10.5772/53423 13

ry of Henryk Wisniewsky in New York by Inge Grundke-Iqbal and colleagues [61].

The nature of islet amyloid polypeptide (IAPP) was discovered by Swedish pathologist Per Westermark from University of Uppsala and his colleagues in 1986 [62,63]. In 1988, apolipo‐ protein A1 (APOA1) was characterized as the amyloid protein in the hereditary amyloid disease in Iowa, USA [64]. In 1990, two groups discovered independently that amyloid fibril protein in the Finnish (AGel) amyloidosis was related to gelsolin [65,66] and identified the causative Asn-187 mutation in the gene for gelsolin [67,68]. The first description of a genetic cause for a hereditary amyloid disease was already published several years earlier, as the Japanese Satoru Tawara and colleagues identified the point mutation in the gene coding for TTR leading to the substitution of methionine instead of valine at position 30 in TTR-related FAP in 1983 [69]. Development of the polymerase chain reaction (PCR) –based techniques have accelerated the identification of mutations in the genes of the amyloidogenic proteins, and today more than one hundred different mutations have been described in the TTR gene. Most of the mutations lead to clinical disease with the deposition of amyloid in different organs.

In the past two decades three different proteins were characterized describing three novel different familial amyloid diseases with a preference for renal manifestation: fibrinogen A-α chain [70], lysozyme [71] and apolipoprotein AII [72]. The last identified amyloid fibril protein, also presenting with renal amyloidosis especially in Mexican Americans, was leucocyte

Characterization of the prion protein (PrP) in 1982 [74] opened up a new perspective in the amyloid reseach. Scrapie, a prion disease in sheeps and cows, was described in Spanish merino sheeps already in 1732 [75] but it took two and a half centuries to detect the causative agent. Scrapie belongs to prion diseases, also referred to "transmissible protein misfolding disorders" [76]. The highest degree in the conformational shift from α-helix to β-sheet structure occurs in

chemotactic factor 2 [73].

**10. Prion diseases**

The "water extraction method" of Pras has been widely used to extract almost all types of amyloid except for Aβ and prion protein amyloid [42,43]. The method was revolutionary in amyloid research as it enabled (1) identification of the β-pleated sheet configuration of amyloid proteins and (2) discovery of the biochemical structure of those proteins.

## **8. The β-pleated sheet configuration**

In nature, most proteins have both α-helix and β-pleated sheet secondary structure. In the amyloid form, the proteins are mostly in the β-pleated sheet conformation though not exclusively. Factors that may influence changes in the spatial form of proteins include increased protein content, low pH, metal ions proteins that are associated with amyloid deposits but are not part of the insoluble fibrils themselves, also called " chaperones" [44]. The secondary structure of amyloid consists of the polypeptide backbone, mostly in the β-pleated sheet conformation, oriented perpendicular to the fibril axis. This β-pleated sheet structure was revealed by X-ray diffraction analysis of isolated amyloid protein fibrils by Eanes and Glenner in 1968 [45-47].

## **9. Identification of the different amyloid proteins**

The major consequence of the invention of the water extraction method of Pras was the identification of the biochemical composition of several kinds of amyloids. Glenner and his colleagues soon applied the method to the "primary" (today: AL) amyloidosis [48] and found the relationship between this amyloidosis and immunoglobulin light chains. AL amyloidosis is a neoplasticdisease andbelongs to the clinicallymost significant amyloid-relatedconditions.

During the subsequent years, a long list of amyloid proteins was identified one after another. Inflammation-associated amyloidosis, previously called the "secondary" and today AA amyloidosis, was shown to be caused by amyloid protein A, an acute phase protein in 1972 [49]. Serum proteinA was thereafter soon identifiedin blood[50,51].In 1978,prealbumin (transthyr‐ etin, TTR) was found to be the protein constituent of amyloid deposits in Portuguese familial amyloid polyneuropathy (FAP) [52], the clinical condition having been described already in 1951 (Corino de Andrade, M. Preliminary note on an unusual form of peripheral neuropathy. Rev Neurol (Paris) 85: 302-6, 1951; cited in Kyle [2]). Similar diseases were found especially in Japan and Sweden in the subsequent decades. The Finnish type of familial amyloidosis, today known as AGel amyloidosis, was described in 1969 by the Finnish ophthalmologist Jouko Meretoja [53]. In 1980, TTR was characterized as the amyloid protein also in "senile cardiac amyloidosis" (SCA) [54], later renamed as senile systemic amyloidosis (SSA) [55,56]. In 1983, the Icelandic type of familial cerebral amyloid angiopathy (HCHWA-I) was found to be related to cystatin-C protein [57]. Next year, 1984, the first report on the AD-associated Aβ protein was published two Glenner and Wong who identified Aβ in the cerebrovascular tissue [58]. Colin Masters (Australian), Konrad Beyreuther (German) and colleagues described the same protein one year later, in 1985, in the plaques (Figure 3B). Japanese Fumitage Gejyo described beta 2 microglobulin as the amyloid fibril protein in the dialysis-related amyloid arthropathy in the same year [60]. Tau protein of the neurofibrillary tangles was identified in 1986 in the laborato‐ ry of Henryk Wisniewsky in New York by Inge Grundke-Iqbal and colleagues [61].

The nature of islet amyloid polypeptide (IAPP) was discovered by Swedish pathologist Per Westermark from University of Uppsala and his colleagues in 1986 [62,63]. In 1988, apolipo‐ protein A1 (APOA1) was characterized as the amyloid protein in the hereditary amyloid disease in Iowa, USA [64]. In 1990, two groups discovered independently that amyloid fibril protein in the Finnish (AGel) amyloidosis was related to gelsolin [65,66] and identified the causative Asn-187 mutation in the gene for gelsolin [67,68]. The first description of a genetic cause for a hereditary amyloid disease was already published several years earlier, as the Japanese Satoru Tawara and colleagues identified the point mutation in the gene coding for TTR leading to the substitution of methionine instead of valine at position 30 in TTR-related FAP in 1983 [69]. Development of the polymerase chain reaction (PCR) –based techniques have accelerated the identification of mutations in the genes of the amyloidogenic proteins, and today more than one hundred different mutations have been described in the TTR gene. Most of the mutations lead to clinical disease with the deposition of amyloid in different organs.

In the past two decades three different proteins were characterized describing three novel different familial amyloid diseases with a preference for renal manifestation: fibrinogen A-α chain [70], lysozyme [71] and apolipoprotein AII [72]. The last identified amyloid fibril protein, also presenting with renal amyloidosis especially in Mexican Americans, was leucocyte chemotactic factor 2 [73].

## **10. Prion diseases**

A significant step forward took place when M Pras (originally from the Tel Hashomer Hospital, Tel Aviv, Israel) and colleagues from New York University School of Medicine described the method to extract proteins from amyloid-laden tissues using water [41]. Spleen tissue from a deceased patient with "primary" (i.e. AA) amyloidosis was homogenized with physiological saline (NaCl), and the mixture was centrifuged. The sediment was next homogenized with NaCl several times to remove most of the soluble proteins and other soluble materials. Salt was then removed by homogenizing the residue in distilled water, followed by centrifugation of the suspension. Lastly, the residue was homogenized in distilled water and centrifuged four times to give a supernatant rich in protein. Adding Congo red dye and NaCl then resulted in a gelatinous precipitate with the typical green birefringence, demonstrating that the superna‐

The "water extraction method" of Pras has been widely used to extract almost all types of amyloid except for Aβ and prion protein amyloid [42,43]. The method was revolutionary in amyloid research as it enabled (1) identification of the β-pleated sheet configuration of amyloid

In nature, most proteins have both α-helix and β-pleated sheet secondary structure. In the amyloid form, the proteins are mostly in the β-pleated sheet conformation though not exclusively. Factors that may influence changes in the spatial form of proteins include increased protein content, low pH, metal ions proteins that are associated with amyloid deposits but are not part of the insoluble fibrils themselves, also called " chaperones" [44]. The secondary structure of amyloid consists of the polypeptide backbone, mostly in the β-pleated sheet conformation, oriented perpendicular to the fibril axis. This β-pleated sheet structure was revealed by X-ray diffraction analysis of isolated amyloid protein fibrils by Eanes and

The major consequence of the invention of the water extraction method of Pras was the identification of the biochemical composition of several kinds of amyloids. Glenner and his colleagues soon applied the method to the "primary" (today: AL) amyloidosis [48] and found the relationship between this amyloidosis and immunoglobulin light chains. AL amyloidosis is a neoplasticdisease andbelongs to the clinicallymost significant amyloid-relatedconditions. During the subsequent years, a long list of amyloid proteins was identified one after another. Inflammation-associated amyloidosis, previously called the "secondary" and today AA amyloidosis, was shown to be caused by amyloid protein A, an acute phase protein in 1972 [49]. Serum proteinA was thereafter soon identifiedin blood[50,51].In 1978,prealbumin (transthyr‐ etin, TTR) was found to be the protein constituent of amyloid deposits in Portuguese familial

proteins and (2) discovery of the biochemical structure of those proteins.

**9. Identification of the different amyloid proteins**

tant represented soluble amyloid.

12 Amyloidosis

Glenner in 1968 [45-47].

**8. The β-pleated sheet configuration**

Characterization of the prion protein (PrP) in 1982 [74] opened up a new perspective in the amyloid reseach. Scrapie, a prion disease in sheeps and cows, was described in Spanish merino sheeps already in 1732 [75] but it took two and a half centuries to detect the causative agent. Scrapie belongs to prion diseases, also referred to "transmissible protein misfolding disorders" [76]. The highest degree in the conformational shift from α-helix to β-sheet structure occurs in the genetically determined form (Gerstmann-Straussler- Scheinken disease) and in PrP associated CAA [77].

**Amyloid protein**

**Precursor protein Type Syndrome (Involved tissue)**

AA (Apo)serum AA S Reactive, previously: "secondary"

AApoAI Apolipoprotein AI S, L Familial (aorta, meniscus)

ABri ABriPP S Familial dementia, British ACal (Pro)calcitonin L C-cell thyroid tumors

ADan ADanPP L Familial dementia, Danish

AGel Gelsolin S Familial, previously: "Finnish"

AH Immunoglobulin heavy chain S; L Myeloma-associated, previously: "primary" AIAPP Islet amyloid polypeptide L Insulinomas, aging, previously: "amylin" (Islets

AL Immunoglobulin light chain S; L Myeloma-associated, previously: "primary"

AMed Lactadherin L Aortic and arterial media, aging

ASemI Semenogelin I L (Vesicula seminalis)

Aβ Aβ protein precursor (AβPP) L Aging, AD, CAA

APro Prolactin L Prolactinomas, aging (pituitary gland) APrP Prion protein L Spongiform encephalopathies (brain)

ATTR Transthyretin S,L? Familial, SSA (localized: tenosynovium)

Aβ2M β2-microglobulin S; L? Hemodialysis-associated (localized: joints) S = systemic; L = localized; SSA = senile systemic amyloidosis; AD = Alzheimer's disease; CAA = cerebral amyloid

of Langerhans)

"Amyloid" — Historical Aspects http://dx.doi.org/10.5772/53423 15

L Odonogenic tumors

AANF Atrial natriuretic factor L (Cardiac atria)

AApoAIV Apolipoprotein AIV S Sporadic, aging

AApoAII Apolipoprotein AII S Familial

ACys Cystatin C S Familial

AFib Fibrinogen α-chain S Familial

AIns Insulin L Iatrogenic AKer Kerato-epithelin L Familial (cornea)

ALac Lactoferrin L (Cornea) ALect2 Leukocyte chemotactic factor 2 S Mainly kidney ALys Lyspzyme S Familial

AOAAP Odontogenic ameloblast-associated

**Table 1.** Human amyloid fibril proteins and their precursors.

protein

angiopathy.

Prion diseases occur in several mammalian species and can be sporadic, hereditary, or acquired. The disease exists in nine different types in humans. The first known descriptions of human prion disease appeared independently in 1920 and 1921 by Creuzfeld and (Creutz‐ feldt HG: Über eine eigenartige herdformige erkrankung des Zentralnervensystems. Z gesamte Neurol Psychiatr 1920, 57: 1-19) and Jacob (Jacob A: Über eigenartige Erkrankungen des Zentralnervensystems mit bemerkenswertem anatomischen Befunde. (Spastische Pseu‐ doskleros- Encephalomyelopathie mit disseminierten Degenerationsherden). Z Gesamte Neurol Psychiatr 1921, 64: 147-228.), cited in Imram [76]). This has formed the basis for the contemporary name of the disease: "sporadic Creutzfeldt –Jacob's disease".

Two Australian anthropologists, Ronald and Catherine Berndt, were the first to describe the peculiar disease occurring in the Fore linguistic group of people in the Australian Pretectorate of New Guinea, today Papua-New Guinea. Vincent Zigas, the district medical officer started to study the disease in 1957 with the young American virologist and pediatrician Carleton Gajdusek who was interested in infectious diseases. The clinical picture of the disease, as described in the article: "Degenerative Disease of the Central Nervous System in New Guinea — The Endemic Occurrence of Kuru in the Native Population" [78], cited in Libersky [79], consisted of headache and pain, cerebellar ataxia, tremors, shivering and choreiform or athetoid movements. The disease, named as "kuru", occurred exclusively in that Fore linguistic group people and was due to the ritualistic cannibalism ("transumption"). Kuru was neuro‐ pathologically characterized with neuronal degeneration, myelin degeneration, astroglial and microglial proliferation and plaque formations [80]. Interestingly, another human prion disease presenting with similar plaques occurred in Western Europe four decades later. The disease, first reported in UK in 1996 by British investigators and called as "variant CJD" [81] however manifested differently. The typical features of variant CJD include agitation, aggres‐ sion, apathy and paranoid delusions [81]. BSE (Bovine spongiform encephalopathy) prions were soon shown to be causally linked with variant CJD [82].

The American scientist Stanley Prusiner purified the prion protein (PrP Scr) from sheep in 1982 [83].Thename "prion"wasbasedonthe letters oftheword*Pro*te*in*aceus.Prusiner assumedthat PrP wouldact solely in theprotein level withoutinfluence of any genetic material, as it hadbeen proposed several years previously [84]. This and several other several issues are still open, such as if there are factors rendering cells capable of replicating prions and propagating them to the nervous system, and if PrP is fully infective without any cofactors [85]. Gajdusek (in 1976) and Prusiner (in 1997) were honored with Nobel Prize in Medicine for their work in prion diseases.

## **11. Nomenclature**

The modern nomenclature of different types of amyloids (Table 1) is based on the amyloid fibril protein. An originally informal amyloid nomenclature committee was established in 1974


S = systemic; L = localized; SSA = senile systemic amyloidosis; AD = Alzheimer's disease; CAA = cerebral amyloid angiopathy.

**Table 1.** Human amyloid fibril proteins and their precursors.

the genetically determined form (Gerstmann-Straussler- Scheinken disease) and in PrP -

Prion diseases occur in several mammalian species and can be sporadic, hereditary, or acquired. The disease exists in nine different types in humans. The first known descriptions of human prion disease appeared independently in 1920 and 1921 by Creuzfeld and (Creutz‐ feldt HG: Über eine eigenartige herdformige erkrankung des Zentralnervensystems. Z gesamte Neurol Psychiatr 1920, 57: 1-19) and Jacob (Jacob A: Über eigenartige Erkrankungen des Zentralnervensystems mit bemerkenswertem anatomischen Befunde. (Spastische Pseu‐ doskleros- Encephalomyelopathie mit disseminierten Degenerationsherden). Z Gesamte Neurol Psychiatr 1921, 64: 147-228.), cited in Imram [76]). This has formed the basis for the

Two Australian anthropologists, Ronald and Catherine Berndt, were the first to describe the peculiar disease occurring in the Fore linguistic group of people in the Australian Pretectorate of New Guinea, today Papua-New Guinea. Vincent Zigas, the district medical officer started to study the disease in 1957 with the young American virologist and pediatrician Carleton Gajdusek who was interested in infectious diseases. The clinical picture of the disease, as described in the article: "Degenerative Disease of the Central Nervous System in New Guinea — The Endemic Occurrence of Kuru in the Native Population" [78], cited in Libersky [79], consisted of headache and pain, cerebellar ataxia, tremors, shivering and choreiform or athetoid movements. The disease, named as "kuru", occurred exclusively in that Fore linguistic group people and was due to the ritualistic cannibalism ("transumption"). Kuru was neuro‐ pathologically characterized with neuronal degeneration, myelin degeneration, astroglial and microglial proliferation and plaque formations [80]. Interestingly, another human prion disease presenting with similar plaques occurred in Western Europe four decades later. The disease, first reported in UK in 1996 by British investigators and called as "variant CJD" [81] however manifested differently. The typical features of variant CJD include agitation, aggres‐ sion, apathy and paranoid delusions [81]. BSE (Bovine spongiform encephalopathy) prions

The American scientist Stanley Prusiner purified the prion protein (PrP Scr) from sheep in 1982 [83].Thename "prion"wasbasedonthe letters oftheword*Pro*te*in*aceus.Prusiner assumedthat PrP wouldact solely in theprotein level withoutinfluence of any genetic material, as it hadbeen proposed several years previously [84]. This and several other several issues are still open, such as if there are factors rendering cells capable of replicating prions and propagating them to the nervous system, and if PrP is fully infective without any cofactors [85]. Gajdusek (in 1976) and Prusiner (in 1997) were honored with Nobel Prize in Medicine for their work in prion diseases.

The modern nomenclature of different types of amyloids (Table 1) is based on the amyloid fibril protein. An originally informal amyloid nomenclature committee was established in 1974

contemporary name of the disease: "sporadic Creutzfeldt –Jacob's disease".

were soon shown to be causally linked with variant CJD [82].

associated CAA [77].

14 Amyloidosis

**11. Nomenclature**

in Helsinki, Finland, in connection with the 1st International Symposium on Amyloidosis. The 1st official nomenclature committee was founded at the 3rd international symposium on amyloidosis in Povoa de Varzim, Portugal (1979). Thereafter the committee (the Nomenclature Committee of the International Society of Amyloidosis) has met several times to create the official nomenclature lists for each types of amyloid. The last meeting took place in 2010 in Rome, Italy (2010), in conjunction with the 12th International Symposium on Amyloidosis [86]. The reports of the meetings are published in "Amyloid", the official journal of amyloid diseases.

Virchow so greatly are mostly composed of glycogen-like substances with sulfate and phosphate groups. In this regard, Virchow actually was right. On the other hand, it has also turned out that those structures do not represent amyloid. Therefore, it can be asked why the term "amyloid" still has prevailed. The most apparent explanation is Virchow's standing as one of the most valued scientists of his time and probably also the iodine staining that was

"Amyloid" — Historical Aspects http://dx.doi.org/10.5772/53423 17

Amyloid research has traditionally related to the diagnosis and clinical manifestations of the deposition of amyloid in the tissues and organs in diverse disease conditions. Applications in other branches of science such as biotechnology may outline the future prospects in the

Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsin‐

[1] Kumar V, Abbas AK, Fausto N, Mitchell RN. Robbins Basic Pathology, 8th edition.

[2] Kyle RA. Amyloidosis: a convoluted story. Br J Haematol 2001;114(3) 529-538.

[3] Matthias Jacob Scleiden, Theodor Schwann, Max Schulze. Klassische Schriften zur Zellenlehre. In: Ostwalds klassiker der exakten Wissenschaften, band 275: Verlag Harri Deutsch. Available from http//books.google/fi/books/about/Klassische\_Schrif‐

[4] Aterman K. A historical note on the iodine-sulphuric acid reaction of amyloid. Histo‐

[5] Dr. J. M. Schleiden. Scientific botany. First book: chemistry of Plants, Chapter II of the organic elements, section I of the assimilated bodies, p9. Available from htpp:ar‐

[6] Vasil IK. A history of plant biotechnology: from the Cell Theory of Schleiden and Schwann to biotech crops. Plant Cell Rep 2008;27(9) 1423-1440. Doi:10.1007/

used for a long time as the diagnostic test for amyloid [93].

Address all correspondence to: maarit.tanskanen@helsinki.fi

Philadelphia, USA: Saunders; 2007. p166.

ten\_zur\_Zellenlehre.html?=h9\_WFhLD8uYC&redir\_esc=y.

amyloid field [94].

**Author details**

Maarit Tanskanen

ki, Finland

**References**

Doi:bjh2999 [pii].

s00299-008-0571-4.

chemistry 1976;49(2) 131-143.

chive.org/details/priciplesofscie00schlrich.

To be included in the official nomenclature list, the amyloid fibril protein fibril must have been unambiguosly characterized and described in a peer-reviewed journal. The present nomen‐ clature list contains 27 fibril proteins capable to cause human disease. Nine of them have been tested in animals. There are also at least and six proteins appearing as *intracellular* inclusions, with all or some properties of amyloid [86].

## **12. The clinical diagnosis of amyloidosis**

The diagnosis of the depositon of amyloid in diverse clinical conditions has traditionally needed a tissue sample stained with Congo red or thioflavin compounds, followed by the definition of the fibril protein using the IHC-based techniques. These techniques, using commercially available antibodies are quite well applicable in most of the clinically significant amyloid diseases.

The usage of radiological techniques to detect amyloid deposits started in 1988 when Philip Hawkins (London) reported on the usage of in vivo radiological techniques using the 123I labeled serum amyloid P component (SAP) in mice [87]. Two years later the same technique was applied successfully in humans [88]. Recently, antibodies to human SAP molecule were even shown to have potential therapeutic properties in both mice and humans [89], based on the ability of the antibodies to trigger a giant-cell reaction to eliminate visceral amyloid deposits. Another milestone in the radiological diagnostics of amyloid diseases was the discovery by Klunk and colleagues (University of Pittsburgh, US) of the 11C-labeled PET tracer "Pittsburgh compound B" (PiB) to bind selectively to fibrillar Aβ [90]. This made it possible to reveal amyloid pathology *noninvasively* in subjects with AD pathology. Yet, the half-time (T1/2) of 11C is very short (about 20 min) and therefore not applicable in clinical use. The recent invention of a comparable 18F-labeled tracer with much longer T1/2 (110 min) is expected to expand the applicability of PET in a larger number of patients. Of the potential 18F-labeled tracers tested, 18F-AV-45[91] seems to be the most promising [92].

### **13. Conclusion**

The concept of amyloid has transformed several times during the nearly two century long research history of the issue. It is now clear that the cerebral corpora amylacea that inspired Virchow so greatly are mostly composed of glycogen-like substances with sulfate and phosphate groups. In this regard, Virchow actually was right. On the other hand, it has also turned out that those structures do not represent amyloid. Therefore, it can be asked why the term "amyloid" still has prevailed. The most apparent explanation is Virchow's standing as one of the most valued scientists of his time and probably also the iodine staining that was used for a long time as the diagnostic test for amyloid [93].

Amyloid research has traditionally related to the diagnosis and clinical manifestations of the deposition of amyloid in the tissues and organs in diverse disease conditions. Applications in other branches of science such as biotechnology may outline the future prospects in the amyloid field [94].

## **Author details**

in Helsinki, Finland, in connection with the 1st International Symposium on Amyloidosis. The 1st official nomenclature committee was founded at the 3rd international symposium on amyloidosis in Povoa de Varzim, Portugal (1979). Thereafter the committee (the Nomenclature Committee of the International Society of Amyloidosis) has met several times to create the official nomenclature lists for each types of amyloid. The last meeting took place in 2010 in Rome, Italy (2010), in conjunction with the 12th International Symposium on Amyloidosis [86]. The reports of the meetings are published in "Amyloid", the official journal of amyloid

To be included in the official nomenclature list, the amyloid fibril protein fibril must have been unambiguosly characterized and described in a peer-reviewed journal. The present nomen‐ clature list contains 27 fibril proteins capable to cause human disease. Nine of them have been tested in animals. There are also at least and six proteins appearing as *intracellular* inclusions,

The diagnosis of the depositon of amyloid in diverse clinical conditions has traditionally needed a tissue sample stained with Congo red or thioflavin compounds, followed by the definition of the fibril protein using the IHC-based techniques. These techniques, using commercially available antibodies are quite well applicable in most of the clinically significant

The usage of radiological techniques to detect amyloid deposits started in 1988 when Philip Hawkins (London) reported on the usage of in vivo radiological techniques using the 123I labeled serum amyloid P component (SAP) in mice [87]. Two years later the same technique was applied successfully in humans [88]. Recently, antibodies to human SAP molecule were even shown to have potential therapeutic properties in both mice and humans [89], based on the ability of the antibodies to trigger a giant-cell reaction to eliminate visceral amyloid deposits. Another milestone in the radiological diagnostics of amyloid diseases was the discovery by Klunk and colleagues (University of Pittsburgh, US) of the 11C-labeled PET tracer "Pittsburgh compound B" (PiB) to bind selectively to fibrillar Aβ [90]. This made it possible to reveal amyloid pathology *noninvasively* in subjects with AD pathology. Yet, the half-time (T1/2) of 11C is very short (about 20 min) and therefore not applicable in clinical use. The recent invention of a comparable 18F-labeled tracer with much longer T1/2 (110 min) is expected to expand the applicability of PET in a larger number of patients. Of the potential 18F-labeled

The concept of amyloid has transformed several times during the nearly two century long research history of the issue. It is now clear that the cerebral corpora amylacea that inspired

diseases.

16 Amyloidosis

amyloid diseases.

**13. Conclusion**

with all or some properties of amyloid [86].

**12. The clinical diagnosis of amyloidosis**

tracers tested, 18F-AV-45[91] seems to be the most promising [92].

Maarit Tanskanen

Address all correspondence to: maarit.tanskanen@helsinki.fi

Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, Helsin‐ ki, Finland

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22 Amyloidosis


[91] Choi SR, Golding G, Zhuang Z, Zhang W, Lim N, Hefti F et al. Preclinical properties of 18F-AV-45: a PET agent for Abeta plaques in the brain. J Nucl Med 2009;50(11) 1887-1894. Doi:10.2967/jnumed.109.065284.

**Chapter 2**

**Diagnosis of Amyloidosis**

Cezar Augusto Muniz Caldas and

Additional information is available at the end of the chapter

Despite a strong clinical suspicion of amyloidosis, the diagnosis must be confirmed by tissue biopsy. Histological examination of biopsy specimens demonstrates an amorphous, eosino‐ philic substance that stains pink with the Congo red, and displays characteristic apple-green birefringence by polarized microscopy [1]. The histological analysis is the only method for es‐ tablishing the diagnosis of amyloidosis [2,3]. The deposition of amyloid occurs in extracellular matrix, and often in a perivascular distribution with some degree of heterogeneity [1-3].

Although in the systemic amyloidosis the biopsies can be obtained from any organ affected, the blood vessel fragility associated with amyloid deposition carries a risk of bleeding [2,3]. Thus, in the clinical routine, biopsies from non-symptomatic sites are more commonly used [2,3]. In the past, rectal and gingival biopsies were considered the gold standard for the di‐ agnosis of amyloidosis, but actually, abdominal fat pad aspiration has been the preferred

Westermark and Stenkvist in 1973 described a method to remove pieces of subcutaneous ab‐ dominal fat for diagnosis of amyloidosis[3].Although some variants has been described, nor‐ mally the aspiration is done using an 18-23 gauge needle, with 2-5 aspirations [3,5]. The sensitivity reported range from 55-75% and specificity is over than 90% [2,6]. Guy and Jones, analyzing the performance of the abdominal fat pad aspiration in 45 patients with systemic amyloidosis found sensitivity of 58%, specificity of 100%, positive predictive value of 100% and negative predictive value of 85%, confirming the accuracy of the methodology [7].

and reproduction in any medium, provided the original work is properly cited.

© 2013 Caldas and de Carvalho; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

due its simplicity, low cost, minimal complications, and good accuracy [1,4].

**2. Abdominal fat pad aspiration or biopsy**

Jozélio Freire de Carvalho

http://dx.doi.org/10.5772/52901

**1. Introduction**


**Chapter 2**

## **Diagnosis of Amyloidosis**

Cezar Augusto Muniz Caldas and Jozélio Freire de Carvalho

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52901

## **1. Introduction**

[91] Choi SR, Golding G, Zhuang Z, Zhang W, Lim N, Hefti F et al. Preclinical properties of 18F-AV-45: a PET agent for Abeta plaques in the brain. J Nucl Med 2009;50(11)

[92] Furst AJ, Kerchner GA. From Alois to Amyvid: Seeing Alzheimer disease. Neurology

[93] Doyle L. Lardaceous disease: some early reports by British authors (1722-1879). J R

[94] Sweers KK, Bennink ML, Subramaniam V. Nanomechanical properties of single amyloid fibrils. J Phys Condens Matter 2012;24(24) 243101. Doi:

1887-1894. Doi:10.2967/jnumed.109.065284.

2012. Doi:10.1212/WNL.0b013e3182662084.

Soc Med 1988;81(12) 729-731.

24 Amyloidosis

10.1088/0953-8984/24/24/243101.

Despite a strong clinical suspicion of amyloidosis, the diagnosis must be confirmed by tissue biopsy. Histological examination of biopsy specimens demonstrates an amorphous, eosino‐ philic substance that stains pink with the Congo red, and displays characteristic apple-green birefringence by polarized microscopy [1]. The histological analysis is the only method for es‐ tablishing the diagnosis of amyloidosis [2,3]. The deposition of amyloid occurs in extracellular matrix, and often in a perivascular distribution with some degree of heterogeneity [1-3].

Although in the systemic amyloidosis the biopsies can be obtained from any organ affected, the blood vessel fragility associated with amyloid deposition carries a risk of bleeding [2,3]. Thus, in the clinical routine, biopsies from non-symptomatic sites are more commonly used [2,3]. In the past, rectal and gingival biopsies were considered the gold standard for the di‐ agnosis of amyloidosis, but actually, abdominal fat pad aspiration has been the preferred due its simplicity, low cost, minimal complications, and good accuracy [1,4].

## **2. Abdominal fat pad aspiration or biopsy**

Westermark and Stenkvist in 1973 described a method to remove pieces of subcutaneous ab‐ dominal fat for diagnosis of amyloidosis[3].Although some variants has been described, nor‐ mally the aspiration is done using an 18-23 gauge needle, with 2-5 aspirations [3,5]. The sensitivity reported range from 55-75% and specificity is over than 90% [2,6]. Guy and Jones, analyzing the performance of the abdominal fat pad aspiration in 45 patients with systemic amyloidosis found sensitivity of 58%, specificity of 100%, positive predictive value of 100% and negative predictive value of 85%, confirming the accuracy of the methodology [7].

© 2013 Caldas and de Carvalho; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The clinician and pathologist must be familiarized with the methodology, histological pit‐ falls and the possibility of false negative, as possible in preferential deposition in terms of organ involvement of amyloid depending of its subtype, as the transthyretin type, with its predilection to deposit in the heart [1,3]. Another situation that can result in false negative, for example, is when the disease is an early stage with amyloid deposits in plaques [8].

the duodenum, 83% in the rectum, and 29% in the gingiva, without complications related to

Diagnosis of Amyloidosis http://dx.doi.org/10.5772/52901 27

Since the cardiac involvement is the major prognostic determinant in systemic amyloidosis [16], the evidence of cardiac lesion is crucial to therapeutic decisions. The gold standard test for diagnosing cardiac amyloidosis is the endomyocardial biopsy, however, it is not performed routinely due risk of complications, although infrequent, such as ventricular wall perforation, cardiac tamponade, pneumothorax, and arrhythmias [17]. Therefore, the cardiac amyloidosis is normally established by echocardiographic evidence of amyloidosis and histologic confir‐ mation of amyloid on noncardiac tissue [17]. The changes observed in the echocardiography are those of restrictive cardiomyopathy with concentric ventricular hypertrophy, especially in the interventricular septum and posterior wall of the left ventricle [3]. Low voltage on electro‐ cardiography and interventricularseptal thickness of > 19.8mm on echocardiography together

Effective medical treatment needs an accurate diagnosis with demonstration of amyloid deposition in the tissues and accurate molecular classification of amyloidosis [1,19,20]. For example, in AL amyloidosis, derived from immunoglobulin light chain, the cornerstone of treatment is the aggressive treatment of the underlying neoplastic process, and in AA amy‐

Immunohistochemistry is currently the standard methodology for amyloid typing in routine clinical practice; it has been able to identify amyloid deposits through binding antibodies di‐ rected against most of the amyloid molecules identified to date. In patients with systemic amyloidosis, studies with antibodies to AA and to the immunoglobulin light chains are usu‐ ally sufficient [2,20]. Some pitfalls are present in the clinical practice, and in some cases, mis‐ diagnoses may occur, especially when immunohistochemical staining is performed in the

The majority of cases of AA can be reliably typed in frozen and/or paraffin sections, but immu‐ nohistochemical typing of AL is still challenging, due commercial antibodies are raised against the constant regions of the respective immunoglobulin light chains, and whether a subset of AL, in which amyloid fibrils are derived from a truncated light chain (ie, containing only vari‐

Another important pitfall is the presence of background stainin the tissue, which in paraffin sections in particular can be significant due the "locking-in" of serum proteins during fixation [20]. The use of frozen specimens and immunofluorescence stains considerably increases the reliability and reproducibility of labeling with antibodies to immunoglobulin light chain, due provide a cleaner background [20,28]. Picken emphasizes that the interpretation of immuno‐ histochemistry performed in paraffin sections and immunofluorescence in frozen sections is not a simple matter and also depends on the experience and expertise of the operator [20].

able regions), will be expected to be nonreactive with commercial antibodies [25-27].

have a sensitivity of 72% and specificity of 91% for cardiac amyloidosis [18].

loidosis, the target of treatment is the underlying inflammatory disease [20-23].

absence of standardized antibodies and appropriate positive controls [24].

**5. Determining the type of amyloid protein**

endoscopy or biopsies [15].

## **3. Rectal biopsy and others gastrointestinal tract sites**

The rectal biopsy was the most used diagnostic method in the past. Actually it has been re‐ placed by abdominal fat pad aspiration, because this is more feasible in the clinical practice with low cost and lack of complications. Analysis of deep fragments including the submuco‐ sa, obtained during a rectoscopic examination, the sensitivity ranges from 75-85% [3,9].

Other sites of gastrointestinal tract can be biopsied. Tada studied 42 patients with gastroin‐ testinal amyloidosis and found amyloid deposition especially in the duodenum and jejunum [10]. Okuda Y et al had similar results assessing rheumatoid arthritis patients, where the proportion of amyloid deposition was 76.5% for duodenal cap and 88.6% for second portion of the duodenum, suggesting a good efficacy of duodenal biopsy in this population [11].

Labial and gingival biopsy has been shownuseful in the amyloidosis diagnosis, but the lat‐ eris less sensitivity [3]. Several studies have confirmed the usefulness of labial biopsy, such as Fatihi et al that evaluated labial biopsy in patients with renal amyloidosis and found amyloid deposits in 80% of accessory gland biopsy and 75% of rectal biopsy [12]. Lechapt-Zalcmanet al performed labial salivary biopsy in 32 patients with polyneuropathy of un‐ known origin and detected amyloid deposits in 7 (transthyretin in five and AL in two), proposing this technique as routine in investigation of axonal polyneuropathies [13]. Hachu‐ la et al detected amyloid deposits in 26 of the 30 patients with systemic amyloidosis using labial salivary gland biopsy, emphasizing the importance of this procedure, even in the ab‐ sence of oral symptoms [14].

## **4. Others biopsy sites**

Because there is risk of life-threatening bleeding, biopsy from others sites is used only whether abdominal fat pad aspiration, rectal or labial salivary gland biopsy fail to establish the diagnosis [3].

The kidney is the most frequently involved organ in systemic amyloidosis and although kid‐ ney biopsy is fundamental for diagnosis, this procedure has been contraindicated in some situations, for example, bleeding diathesis, and can be complicated by perirenal hematoma or arteriovenous fistula [15]. Before performing a kidney biopsy, less invasive biopsy proce‐ dures from easily accessible tissues should be considered. Yilmaz M et al studying 78 pa‐ tients with chronic kidney disease found the frequency of amyloid deposition was 100% in the duodenum, 83% in the rectum, and 29% in the gingiva, without complications related to endoscopy or biopsies [15].

Since the cardiac involvement is the major prognostic determinant in systemic amyloidosis [16], the evidence of cardiac lesion is crucial to therapeutic decisions. The gold standard test for diagnosing cardiac amyloidosis is the endomyocardial biopsy, however, it is not performed routinely due risk of complications, although infrequent, such as ventricular wall perforation, cardiac tamponade, pneumothorax, and arrhythmias [17]. Therefore, the cardiac amyloidosis is normally established by echocardiographic evidence of amyloidosis and histologic confir‐ mation of amyloid on noncardiac tissue [17]. The changes observed in the echocardiography are those of restrictive cardiomyopathy with concentric ventricular hypertrophy, especially in the interventricular septum and posterior wall of the left ventricle [3]. Low voltage on electro‐ cardiography and interventricularseptal thickness of > 19.8mm on echocardiography together have a sensitivity of 72% and specificity of 91% for cardiac amyloidosis [18].

## **5. Determining the type of amyloid protein**

The clinician and pathologist must be familiarized with the methodology, histological pit‐ falls and the possibility of false negative, as possible in preferential deposition in terms of organ involvement of amyloid depending of its subtype, as the transthyretin type, with its predilection to deposit in the heart [1,3]. Another situation that can result in false negative, for example, is when the disease is an early stage with amyloid deposits in plaques [8].

The rectal biopsy was the most used diagnostic method in the past. Actually it has been re‐ placed by abdominal fat pad aspiration, because this is more feasible in the clinical practice with low cost and lack of complications. Analysis of deep fragments including the submuco‐ sa, obtained during a rectoscopic examination, the sensitivity ranges from 75-85% [3,9].

Other sites of gastrointestinal tract can be biopsied. Tada studied 42 patients with gastroin‐ testinal amyloidosis and found amyloid deposition especially in the duodenum and jejunum [10]. Okuda Y et al had similar results assessing rheumatoid arthritis patients, where the proportion of amyloid deposition was 76.5% for duodenal cap and 88.6% for second portion of the duodenum, suggesting a good efficacy of duodenal biopsy in this population [11].

Labial and gingival biopsy has been shownuseful in the amyloidosis diagnosis, but the lat‐ eris less sensitivity [3]. Several studies have confirmed the usefulness of labial biopsy, such as Fatihi et al that evaluated labial biopsy in patients with renal amyloidosis and found amyloid deposits in 80% of accessory gland biopsy and 75% of rectal biopsy [12]. Lechapt-Zalcmanet al performed labial salivary biopsy in 32 patients with polyneuropathy of un‐ known origin and detected amyloid deposits in 7 (transthyretin in five and AL in two), proposing this technique as routine in investigation of axonal polyneuropathies [13]. Hachu‐ la et al detected amyloid deposits in 26 of the 30 patients with systemic amyloidosis using labial salivary gland biopsy, emphasizing the importance of this procedure, even in the ab‐

Because there is risk of life-threatening bleeding, biopsy from others sites is used only whether abdominal fat pad aspiration, rectal or labial salivary gland biopsy fail to establish

The kidney is the most frequently involved organ in systemic amyloidosis and although kid‐ ney biopsy is fundamental for diagnosis, this procedure has been contraindicated in some situations, for example, bleeding diathesis, and can be complicated by perirenal hematoma or arteriovenous fistula [15]. Before performing a kidney biopsy, less invasive biopsy proce‐ dures from easily accessible tissues should be considered. Yilmaz M et al studying 78 pa‐ tients with chronic kidney disease found the frequency of amyloid deposition was 100% in

**3. Rectal biopsy and others gastrointestinal tract sites**

sence of oral symptoms [14].

**4. Others biopsy sites**

the diagnosis [3].

26 Amyloidosis

Effective medical treatment needs an accurate diagnosis with demonstration of amyloid deposition in the tissues and accurate molecular classification of amyloidosis [1,19,20]. For example, in AL amyloidosis, derived from immunoglobulin light chain, the cornerstone of treatment is the aggressive treatment of the underlying neoplastic process, and in AA amy‐ loidosis, the target of treatment is the underlying inflammatory disease [20-23].

Immunohistochemistry is currently the standard methodology for amyloid typing in routine clinical practice; it has been able to identify amyloid deposits through binding antibodies di‐ rected against most of the amyloid molecules identified to date. In patients with systemic amyloidosis, studies with antibodies to AA and to the immunoglobulin light chains are usu‐ ally sufficient [2,20]. Some pitfalls are present in the clinical practice, and in some cases, mis‐ diagnoses may occur, especially when immunohistochemical staining is performed in the absence of standardized antibodies and appropriate positive controls [24].

The majority of cases of AA can be reliably typed in frozen and/or paraffin sections, but immu‐ nohistochemical typing of AL is still challenging, due commercial antibodies are raised against the constant regions of the respective immunoglobulin light chains, and whether a subset of AL, in which amyloid fibrils are derived from a truncated light chain (ie, containing only vari‐ able regions), will be expected to be nonreactive with commercial antibodies [25-27].

Another important pitfall is the presence of background stainin the tissue, which in paraffin sections in particular can be significant due the "locking-in" of serum proteins during fixation [20]. The use of frozen specimens and immunofluorescence stains considerably increases the reliability and reproducibility of labeling with antibodies to immunoglobulin light chain, due provide a cleaner background [20,28]. Picken emphasizes that the interpretation of immuno‐ histochemistry performed in paraffin sections and immunofluorescence in frozen sections is not a simple matter and also depends on the experience and expertise of the operator [20].

Since early diagnosis is a very important step to appropriate treatment of transthyretin (TTR) amyloidosis, and this amyloidogenic protein causes two different forms of the disease (hereditary amyloidogenic TTR [ATTR] amyloidosis and senile systemic amyloidosis [SSA]), we should accurately distinguish them. For instance, to detect Val30Met mutation in TTR gene, which is the most frequent pathogenic mutation in hereditary ATTR amyloidosis, some researchers use real-time PCR genotyping assay, considering reliable, rapid, cost-effec‐ tive, and suitable analysis, however, to achieve accurate results the application of both ge‐ netic and proteomic methods is preferable to compensate the disadvantages and possible pitfalls in each of the techniques used [19]. Using proteomics techniques, amyloid typing can be successful in small samples, including biopsies [29,30]. Several TTR variants can be detected in serum specimens using mass spectrometry or sophisticated electrophoresis tech‐ niques [31,32], however, this methodologies, and others new technologies, such as laser mi‐ crodissection, are frequently available only at specialized centers.

**7. Conclusion**

to correct analysis.

**Author details**

**References**

2010;15(6):24.

Affinity: 2009. p388-407.

analysis.J ClinPathol1989;42(8):817-9.

sis.Arch Intern Med1973;132(4):522-3.

538-45.

Cezar Augusto Muniz Caldas1

\*Address all correspondence to: jotafc@gmail.com

Bahia, School of Medicine, Salvador-BA, Brazil

The clinical suspicion must be confirmed with histological examination, and the amyloid typing is crucial to determine the correct treatment. Although the apparently simplicity of the abdominal fat pad aspiration has facilitated the diagnosis of amyloidosis, the physicians should be aware to pitfalls, especially in the amyloid typing, requiring an expert pathologist

Diagnosis of Amyloidosis http://dx.doi.org/10.5772/52901 29

and Jozélio Freire de Carvalho2

1 Internal Medicine Department, Universidade Federal do Pará - UFPA, and Curso de Me‐

2 Rheumatology Division, Hospital Universitário Prof. Edgard Santos, Federal University of

[1] Halloush RA, Lavrovskaya E, Mody DR et al. Diagnosis and typing of systemic amy‐ loidosis: The role of abdominal fat pad fine needle aspiration biopsy. Cytojournal

[2] Hachulla E, Grateau G. Diagnostic tools for amyloidosis. Joint Bone Spine2002;69(6):

[3] Hachulla E, Beyne-Rauzy O, Soubrier M et al. Systemic consequences of the inflam‐ matory process. In: Bijlsma JWJ. (ed.) Eular Compendium on Rheumatic Diseases.

[4] Westermark P, Benson L, Juul J, Sletten K. Use of subcutaneous abdominal fat biopsy specimen for detailed typing of amyloid fibril protein-AL by amino acid sequence

[5] Westermark P, Stenkvist B.A new method for the diagnosis of systemic amyloido‐

[6] Ansari-Lari MA, Ali SZ. Fine-needle aspiration of abdominal fat pad for amyloid de‐

tection: a clinically useful test? DiagnCytopathol2004;30(3): 178-81.

dicina do Centro Universitário do Estado do Pará - CESUPA, Belém-PA, Brazil

## **6. Assessing the extension of involvement in systemic amyloidosis**

The amyloid typing must be followed by distinction between localized and systemic amyloi‐ dosis [20]. While the treatment of localized forms is mainly conservative, the treatment of systemic forms has been more aggressive, and the prognosis is directly related with the dis‐ ease extension, and the organs affected [2,20]. To determine the extension of the disease, some investigations are necessary, and it is presented in Table 1.


ECG – electrocardiography; MRI – magnetic resonance imaging; NT-proBNP – N-terminal pro-brain natriuretic peptide; EMG – electromyography; ACTH – adrenocorticotropic hormone; TSH – thyroid stimulating hormone; PT – prothrombin.

**Table 1.** Determining site and extent of amyloidosis [3,17]

## **7. Conclusion**

Since early diagnosis is a very important step to appropriate treatment of transthyretin (TTR) amyloidosis, and this amyloidogenic protein causes two different forms of the disease (hereditary amyloidogenic TTR [ATTR] amyloidosis and senile systemic amyloidosis [SSA]), we should accurately distinguish them. For instance, to detect Val30Met mutation in TTR gene, which is the most frequent pathogenic mutation in hereditary ATTR amyloidosis, some researchers use real-time PCR genotyping assay, considering reliable, rapid, cost-effec‐ tive, and suitable analysis, however, to achieve accurate results the application of both ge‐ netic and proteomic methods is preferable to compensate the disadvantages and possible pitfalls in each of the techniques used [19]. Using proteomics techniques, amyloid typing can be successful in small samples, including biopsies [29,30]. Several TTR variants can be detected in serum specimens using mass spectrometry or sophisticated electrophoresis tech‐ niques [31,32], however, this methodologies, and others new technologies, such as laser mi‐

crodissection, are frequently available only at specialized centers.

28 Amyloidosis

some investigations are necessary, and it is presented in Table 1.

Liver Liver enzymes Ultrasonography

Eyes Fundoscopy Slit-lamp examination

Nerves - EMG

Endocrine glands ACTH test, TSH -

Haemostasis PT, X factor -

**Table 1.** Determining site and extent of amyloidosis [3,17]

Heart Chest radiography, ECG, echocardiography, MRI, NTproBNP/troponin

**Organ Performed routinely Performed as clinically indicated** Kidneys Proteinuria, serum creatinine, ultrasonography Renal vein Doppler ultrasound

Spleen Ultrasonography, blood cell counts Howell-Jolly bodies in blood smears

**6. Assessing the extension of involvement in systemic amyloidosis**

The amyloid typing must be followed by distinction between localized and systemic amyloi‐ dosis [20]. While the treatment of localized forms is mainly conservative, the treatment of systemic forms has been more aggressive, and the prognosis is directly related with the dis‐ ease extension, and the organs affected [2,20]. To determine the extension of the disease,

Gastrointestinal tract Serum protein electrophoresis Gastrointestinal endoscopy, oesophagealmanometry

Respiratory system Chest radiography Blood gas analysis, bronchoscopy, CT scan of the chest

ECG – electrocardiography; MRI – magnetic resonance imaging; NT-proBNP – N-terminal pro-brain natriuretic peptide; EMG – electromyography; ACTH – adrenocorticotropic hormone; TSH – thyroid stimulating hormone; PT – prothrombin.

99mTc-pyrophosphate scan, 24-h Holter

The clinical suspicion must be confirmed with histological examination, and the amyloid typing is crucial to determine the correct treatment. Although the apparently simplicity of the abdominal fat pad aspiration has facilitated the diagnosis of amyloidosis, the physicians should be aware to pitfalls, especially in the amyloid typing, requiring an expert pathologist to correct analysis.

## **Author details**

Cezar Augusto Muniz Caldas1 and Jozélio Freire de Carvalho2

\*Address all correspondence to: jotafc@gmail.com

1 Internal Medicine Department, Universidade Federal do Pará - UFPA, and Curso de Me‐ dicina do Centro Universitário do Estado do Pará - CESUPA, Belém-PA, Brazil

2 Rheumatology Division, Hospital Universitário Prof. Edgard Santos, Federal University of Bahia, School of Medicine, Salvador-BA, Brazil

## **References**


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[23] Nakamura T.AmyloidA amyloidosis secondary to rheumatoid arthritis: pathophysi‐

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[24] Linke RP, Oos R, Wiegel NM, Nathrath WB. Classification of amyloidosis: misdiag‐ nosing by way of incomplete immunohistochemistry and how to prevent it.ActaHis‐

[25] Picken MM.New insights into systemic amyloidosis: the importance of diagnosis of

[26] Novak L, Cook WJ, Herrera GA, Sanders PW. AL-amyloidosis is underdiagnosed in

[27] Satoskar AA, Burdge K, Cowden DJ et al. Typing of amyloidosis in renal biopsies:

[28] Droz D, Nochy D. Amyloid substance and amyloidosis.Ann Pathol1995;15(1):11-20.

[29] Murphy CL, Wang S, Williams T et al. Characterization of systemic amyloid deposits

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30 Amyloidosis

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**Chapter 3**

**Cardiac Amyloidosis:**

Kyle W. Klarich

**1. Introduction**

http://dx.doi.org/10.5772/53763

**2. Classification of amyloidosis**

Glenn K. Lee, DaLi Feng, Martha Grogan, Cynthia Taub, Angela Dispenzieri and

Additional information is available at the end of the chapter

**Typing, Diagnosis, Prognosis and Management**

Amyloidosis is uncommon, with age-adjusted incidences of between 6.1 and 10.5 per million person-years,[1] and an estimated 1275 to 3200 new cases occurring annually in the United States.[1, 2] The contemporary understanding of amyloidosis points to a group of complex sys‐ temic disorders involving the extracellular deposition of misfolded proteinaceous material in many organs, most commonly the kidneys, heart, liver, central and peripheral nervous sys‐ tems.[2-4] The normal function of tissues is altered, and end-organ dysfunction usually ensues. Cardiac amyloidosis can be isolated to the heart, but it often coexists with disease elsewhere in the body.[4, 5] Cardiac manifestations may predominate the clinical presentation or may be subclinical and detected on routine investigation of a patient presenting with non-cardiac com‐ plaints.[5] The presence and relative prominence of cardiac involvement in the clinical picture is dependent on the type of amyloidosis and severity of amyloid infiltration in the tissue.[5]

Amyloidosis refers to a group of unrelated diseases involving the extracellular deposition of proteinaceous material that demonstrates apple-green birefringence under polarized light on staining with Congo red.[5] In all forms of amyloidosis, abnormal and unstable protein is produced in response to a variety of stimuli and precipitates as amyloid in the extracellular matrix.[2, 3] The contemporary classification of amyloidosis is primarily based on the bio‐ chemistry of the disease process from the precursor amyloid proteins, and comprises several major subgroups. Table 1 describes the typical characteristics of each type of amyloidosis.

> © 2013 Lee et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.
