- Email: [email protected]
Subcutaneous fat tissue for diagnosis and studies of systemic amyloidosis
ARTICLE IN PRESS Acta histochemica 108 (2006) 209—213
www.elsevier.de/acthis
Subcutaneous fat tissue for diagnosis and studies of systemic amyloidosis Per Westermark, Eva Davey, Karolina Lindbom, Stina Enqvist Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, SE-751 85 Uppsala, Sweden
KEYWORDS Subcutaneous fat; Subcutis; Biopsy; Diagnosis; Amyloidosis
Summary The systemic amyloidoses comprise a biochemically heterogeneous group of potentially lethal disorders. An early and precise diagnosis is crucial for the treatment and prognosis. Subcutaneous fat biopsy is a simple and safe method to obtain a diagnosis of systemic amyloidosis and the material can be used for exact determination of amyloid type. A method is described for immunochemical typing of the amyloid based on Western blot analysis combined with specific amyloid fibril protein antibodies. & 2006 Elsevier GmbH. All rights reserved.
Introduction Since the first description of subcutaneous fat tissue biopsy for the diagnosis of amyloidosis (Westermark and Stenkvist, 1971, 1973), initially developed for systemic AA- (secondary amyloidosis), the value and reliability of the method has been confirmed in several studies (Libbey et al., 1983; Orfila et al., 1986; Duston et al., 1987; Maruyama et al., 1987; Gertz et al., 1988; Manganaro et al., 1992; Masouye´, 1997; Guy and Jones, 2001). The method is based on the almost constant involvement of subcutaneous adipose tissue in AA-, AL- and ATTR forms of systemic amyloidosis, and probably in other systemic amyloidoses as well, for example, gelsolin-derived Corresponding author. ,
E-mail address: [email protected] (P. Westermark).
(AGel) amyloidosis (Westermark et al., unpublished result). Amyloid is found both in the walls of small vessels and around the individual fat cells (Schilder, 1909; Westermark, 1972; Orfila et al., 1986). During recent years, the biochemical nature of different forms of systemic amyloidosis and variations in pathogenetic mechanisms have been elucidated. The prognosis depends on the biochemical amyloid forms. Nowadays, there are specific treatments available for some systemic amyloidoses. This new development means that exact and safe determination of the type of amyloid deposit in the individual patient is critical. The more or less constant involvement of subcutaneous adipose tissue makes this an attractive site for biopsy for a biochemical characterization of amyloid material. Not only is the tissue a site of significant amyloid deposits—although rarely causing any symptoms—it also contains comparably low levels of other proteins. Furthermore, a
0065-1281/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2006.03.011
ARTICLE IN PRESS 210 surgical biopsy from abdominal subcutaneous fat tissue is simple and without complications. In many cases it is also possible to purify an amyloid fibril protein from a small subcutaneous tissue biopsy (Westermark et al., 1989; Olsen et al., 1998a). We have used abdominal adipose tissue biopsies for determination of the biochemical type of deposits in systemic amyloidosis for many years. Initially, we used double immunodiffusion as a diagnostic tool. Later, we developed a method based on enzyme-linked immunosorbent assay (ELISA) for amyloid diagnostics (Olsen et al., 1999). For the last 3 years, we have used Western blot analysis for clinical assessment of systemic amyloidosis.
Materials and methods Antisera Since commercially available antibodies often fail in analysis of amyloid fibril proteins, we only use in-house raised antisera, produced in rabbits by standard methods. AL-l antiserum (A-132) was raised against fibril protein, purified from the spleen of an individual with systemic amyloidosis. The protein was determined by N-terminal amino acid sequence analysis to be of l-II type (Olsen et al., 1999). AL k antiserum (A-147) was raised against a synthetic peptide corresponding to a Cterminal part of the constant region of k chains (Olsen et al., 1999). The protein AA antiserum (A126) was also raised against a synthetic peptide corresponding to residues 24–34 of human protein AA (Olsen et al., 1999). Transthyretin amyloid was detected by an antiserum (A1898) raised against an in vitro expressed protein corresponding to residues 50–127 of the human protein (Bergstro ¨m et al., 2004). Antisera against apolipoprotein A-IV (A82/ 01) and apolipoprotein A-I (A-159) were also raised in rabbits against synthetic peptides (Bergstro ¨m et al., 2004; Mucchiano et al., 2001).
P. Westermark et al. specimens are taken from the adipose tissue, squashed between two microscope slides, dried for 5 min and defatted in acetone for 10 min. After evaporation, the slides are stained in Congo red B (Puchtler et al., 1962) solution, diluted 1:10 with Congo red A solution, for 10–20 min. This dilution is important since the specimens otherwise tend to become over-stained with a consequent risk of over-diagnosing amyloidosis (Westermark et al., 1999). The slides are then examined directly for the presence of amyloid deposits, without mounting. The remaining part of the biopsy is defatted overnight in several changes of acetone and airdried. Aliquots are put directly into sample buffer containing 3% sodium dodecyl sulfate (SDS) and 0.02 M dithiothreitol, heated to 100 1C for 30 min and incubated at room temperature overnight. SDS–polyacrylamide gel electrophoresis (PAGE) is performed in a standard Tris–tricine buffer system (Scha ¨gger and von Jagow, 1987) and Western blotting is performed as described by Bergstro ¨m et al. (2004) using an enhanced chemiluminescence system (Amersham Biosciences, Uppsala, Sweden). In each analysis, control amyloid fibrilar materials of known AA, Al, Ak and ATTR are included. The primary antisera are diluted 1:2000, except antiATTR which is diluted 1:10,000.
Results Since we developed the present method, 35 abdominal fat biopsies with amyloid deposits have been analyzed in our laboratory. Of these, 32 have been characterized successfully as detailed in Table 1. AL- and ATTR-amyloid are the commonest types. Whether or not the results are due to the occurrence of amyloid fibrils of two biochemical types is not yet known. A band with mobility of a complete immunoglobulin light chain was often obtained with both antisera against AL-proteins. Such bands were regarded as contaminants from
Method
Table 1. Systemic amyloid forms typed by Western blot analysis of subcutaneous fat biopsies 2002–2004
Subcutaneous fat biopsies (2–20 mm in diameter) are sent to our laboratory in 0.15 M NaCl by ordinary mail. The biopsies are rinsed overnight in 0.15 M NaCl–0.02% sodium azide, followed by rinsing with 0.88% ammonium chloride for hemolysis. This step is important for analysis of purified amyloid fibril proteins, because without it a high background of hemoglobin A and B chains becomes problematic. After washing in distilled water, small
Amyloid type
No. of patients
AL-k AL-l Transthyretin AA AApoA-I AApoA-IV Type not determined
2 12 14 4 0 0 3
ARTICLE IN PRESS Subcutis in systemic amyloidosis
211 cases we obtained a reaction with two of the antisera (AA and ATTR), one with strong bands and the other with weak bands. Biopsies were sent to the laboratory at ambient temperature, without cooling. One advantage of abdominal fat biopsies is their relative robustness and resistance to autolysis and no problems with the quality of the material were noted, even in biopsies which had been in the mail over a weekend.
Discussion
Figure 1. Western blot analysis with antiserum against amyloid protein derived from l type of immunoglobulin light chain. Lanes 2 and 3 contain extracts of fat biopsies from patients with systemic amyloidosis of unknown type. Lane 1 contains control material with amyloid of known AL-l type.
Figure 2. Western blot analysis with antiserum against amyloid protein derived from transthyretin. Lanes 1 and 3 contain extracts of fat biopsies from patients with systemic amyloidosis of unknown type. Lane 2 contains control material with known transthyretin-derived amyloid.
plasma and ignored in the interpretation of the immunoblotting results, which were only based on low molecular protein bands (o20 kDa) (Figs. 1 and 2). In three biopsies, no definite amyloid protein band was detected by Western blot. In these cases, the amount of amyloid was very small, with discrete patches or streaks only in some fat tissue fragments. Usually, the result was clear, but in two
The high proportion of individuals with amyloidosis of TTR-origin may seem surprising, but several biopsies were received from a region in Sweden with a high prevalence of familial amyloidotic polyneuropathy (Suhr et al., 2003). The low proportion of patients with AA-amyloidosis is notable. Most likely, biopsies from this category of individuals are under-represented, probably due to clear diagnosis on clinical grounds since AA-amyloidosis is not a rare condition in Sweden (Lo ¨fberg et al., 1987). One of the difficulties in the development of the amyloid typing method was to obtain antisera which specifically recognized the amyloid fibril proteins. There was no problem with protein AA and it was possible to use several of the tested antisera. The reason that we chose an anti-peptide antibody was that this gave no background labeling and was also useful in immunohistochemistry. The commercial anti-TTR antibody that we tested gave good, specific labeling of TTR monomer and dimer bands, but failed to react with TTR fragments, which constitute most of the amyloid fibrils in many cases of Swedish V30M ATTR-amyloidosis and always in wild-type ATTR disease (senile systemic amyloidosis) (Felding et al., 1985; Westermark et al., 1987). On the other hand, the antiserum against TTR50-127 labeled both full-length and fragmented forms of TTR (Bergstro ¨m et al., in preparation). The antisera against AApoA-I and AApoA-IV amyloid proteins were raised against synthetic peptides, since commercial antisera either did not work (apoA-I) or were not available (apoA-IV). These antisera have been shown to recognize the respective amyloid proteins (Mucchiano et al., 2001; Bergstro ¨m et al., 2004). Finding antisera against AL-proteins was a problem. It is accepted that commercial antisera against immunoglobulin light chains are not reliable for detection of AL-amyloid in immunohistochemistry, including immunofluorescence. Such antisera
ARTICLE IN PRESS 212 are raised against the full-length protein. The amyloid protein is usually an N-terminal fragment of an immunoglobulin light chain, and is most usually unfolded. It may therefore contain exposed epitopes that are not recognized by commercial antisera. Antisera against well-characterized proteins purified from amyloid often exhibit remarkable cross-reactivity, but one antiserum, A-132, was shown to have a high specificity for AL-protein of l type. It recognized all l subtypes tested but not amyloid of AL-k type. The antiserum used for recognition of AL-k protein was raised against a short synthetic peptide corresponding to a segment of k constant region. Although most of the ALamyloid proteins consist of N-terminal fragments of monoclonal light chains, fragments of constant region may be found (Eulitz and Linke, 1985). We demonstrated earlier that small fragments of the Cterminal part of the constant region are always present in amyloid deposits of k type (Olsen et al., 1998b) and the same is true for AL-amyloid of l type (Enqvist et al., in preparation). Our studies have shown that these small fragments can be used for determination of the amyloid type (Olsen et al., 1999). However, it has been difficult to raise a specific antiserum against l constant region, which is effective in the analyses. Several methods for the determination of the type of amyloidosis have been described. These include immunohistochemistry at the light (Linke et al., 1995) and electron microscope level (Arbustini et al., 2002), ELISA (Olsen et al., 1999) and biochemical analysis with Edman degradation and mass spectrometry (Murphy et al., 2001). All these methods work in the right hands. The present method has the advantages that it is technically simple and fast. However, the availability of appropriate specific antibodies is a prerequisite.
Acknowledgments Supported by the Swedish Research Council (Project No. 5941), FAMY, FAMY-Norrbotten and the Amyl Foundation.
References Arbustini E, Verga L, Concardi M, Palladini G, Obici L, Merlini G. Electron and immuno-electron microscopy of abdominal fat identifies and characterizes amyloid fibrils in suspected cardiac amyloidosis. Amyloid 2002;9:108–14. Bergstro ¨m J, Murphy CL, Weiss DT, Solomon A, Sletten K, Hellman U, et al. Two different types of amyloid
P. Westermark et al. deposits – apolipoprotein A-IV and transthyretin – in a patient with systemic amyloidosis. Lab Invest 2004; 84:981–8. Duston MA, Skinner M, Shirahama T, Cohen AS. Diagnosis of amyloidosis by abdominal fat aspiration. Analysis of four years’ experience. Am J Med 1987;82:412–4. Eulitz M, Linke R. Amyloid fibrils derived from V-region together with C-region fragments from a lII-immunoglobulin light chain (HAR). Biol Chem H-S 1985;366: 907–15. Felding P, Fex G, Westermark P, Olofsson B- O, Pitka ¨nen P, Benson L. Prealbumin in Swedish patients with senile systemic amyloidosis and familial amyloidotic polyneuropathy. Scand J Immunol 1985;21:133–40. Gertz MA, Li CY, Shirahama T, Kyle RA. Utility of subcutaneous fat aspiration for the diagnosis of systemic amyloidosis (immunoglobulin light chain). Arch Intern Med 1988;148:929–33. Guy CD, Jones CK. Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls. Diagn Cytopathol 2001;24:181–5. Libbey CA, Skinner M, Cohen AS. Use of abdominal fat tissue aspirate in the diagnosis of systemic amyloidosis. Arch Int Med 1983;143:1549–52. Linke RP, Ga ¨rtner HV, Michels H. High-sensitivity diagnosis of AA amyloidosis using Congo red and immunohistochemistry detects missed amyloid deposits. J Histochem Cytochem 1995;43:863–9. Lo ¨fberg H, Grubb A, Thysell H, Nilsson E, Kjellander B, Moller C, et al. The prevalence of renal amyloidosis of the AA-type in a series of 1158 consecutive autopsies. Acta Pathol Microbiol Immunol Scand [A] 1987;95: 297–302. Manganaro M, Bruno M, Torchio B, Pellerito R, Gabella P, Linari F. Abdominal fat aspiration for diagnosis of amyloidosis. Nephron 1992;60:490–1. Maruyama K, Ikeda S, Yanagisawa N, Nakazato M. Diagnostic value of abdominal fat tissue aspirate in familial amyloidotic polyneuropathy. J Neurol Sci 1987;81:11–8. Masouye´ I. Diagnostic screening of systemic amyloidosis by abdominal fat aspiration: an analysis of 100 cases. Am J Dermatopathol 1997;19:41–5. Mucchiano GI, Ha ¨ggqvist B, Sletten K, Westermark P. Apolipoprotein A-1-derived amyloid in atherosclerotic plaques of the human aorta. J Pathol 2001;193: 270–5. Murphy CL, Eulitz M, Hrncic R, Sletten K, Westermark P, Williams T, et al. Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens. Am J Clin Pathol 2001;116:135–42. Olsen KE, Sletten K, Westermark P. Extended analysis of AL-amyloid protein from abdominal wall subcutaneous fat biopsy: kappa IV immunoglobulin light chain. Biochem Biophys Res Commun 1998a;245:713–6. Olsen KE, Sletten K, Westermark P. Fragments of the constant region of immunoglobulin light chain are constituents of AL-amyloid proteins. Biochem Biophys Res Commun 1998b;251:642–7.
ARTICLE IN PRESS Subcutis in systemic amyloidosis Olsen KE, Sletten K, Westermark P. The use of subcutaneous fat tissue for amyloid typing by enzyme-linked immunosorbent assay. Am J Clin Pathol 1999;111: 355–62. Orfila C, Giraud P, Modesto A, Suc J-M. Abdominal fat tissue aspirate in human amyloidosis: light, electron, and immunofluorescence microscopic studies. Hum Pathol 1986;17:366–9. Puchtler H, Sweat F, Levine M. On the binding of Congo red by amyloid. J Histochem Cytochem 1962;10: 355–64. Scha ¨gger H, von Jagow G. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166:368–79. ¨ ber die amyloide Entartung der Haut. Frankf Schilder P. U Z Pathol 1909;3:782–94. Suhr OB, Svendsen IH, Andersson R, Danielsson A, Holmgren G, Ranlo ¨v PJ. Hereditary transthyretin amyloidosis from a Scandinavian perspective. J Intern Med 2003;254:225–35.
213 Westermark P. Occurrence of amyloid deposits in the skin in secondary systemic amyloidosis. Acta Pathol Microbiol Scand A 1972;80:718–20. Westermark P, Stenkvist B. Diagnosis of secondary generalized amyloidosis by fine needle biopsy of the skin. Acta Med Scand 1971;190:453–4. Westermark P, Stenkvist B. A new method for the diagnosis of systemic amyloidosis. Arch Intern Med 1973;132:522–3. Westermark P, Sletten K, Olofsson B-O. Prealbumin variants in the amyloid fibrils of Swedish familial amyloidotic polyneuropathy. Clin Exp Immunol 1987;69:695–701. 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 analysis. J Clin Pathol 1989;42: 817–9. Westermark GT, Johnson KH, Westermark P. Staining methods for identification of amyloid in tissue. Methods Enzymol 1999;309:3–25.
www.elsevier.de/acthis
Subcutaneous fat tissue for diagnosis and studies of systemic amyloidosis Per Westermark, Eva Davey, Karolina Lindbom, Stina Enqvist Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, SE-751 85 Uppsala, Sweden
KEYWORDS Subcutaneous fat; Subcutis; Biopsy; Diagnosis; Amyloidosis
Summary The systemic amyloidoses comprise a biochemically heterogeneous group of potentially lethal disorders. An early and precise diagnosis is crucial for the treatment and prognosis. Subcutaneous fat biopsy is a simple and safe method to obtain a diagnosis of systemic amyloidosis and the material can be used for exact determination of amyloid type. A method is described for immunochemical typing of the amyloid based on Western blot analysis combined with specific amyloid fibril protein antibodies. & 2006 Elsevier GmbH. All rights reserved.
Introduction Since the first description of subcutaneous fat tissue biopsy for the diagnosis of amyloidosis (Westermark and Stenkvist, 1971, 1973), initially developed for systemic AA- (secondary amyloidosis), the value and reliability of the method has been confirmed in several studies (Libbey et al., 1983; Orfila et al., 1986; Duston et al., 1987; Maruyama et al., 1987; Gertz et al., 1988; Manganaro et al., 1992; Masouye´, 1997; Guy and Jones, 2001). The method is based on the almost constant involvement of subcutaneous adipose tissue in AA-, AL- and ATTR forms of systemic amyloidosis, and probably in other systemic amyloidoses as well, for example, gelsolin-derived Corresponding author. ,
E-mail address: [email protected] (P. Westermark).
(AGel) amyloidosis (Westermark et al., unpublished result). Amyloid is found both in the walls of small vessels and around the individual fat cells (Schilder, 1909; Westermark, 1972; Orfila et al., 1986). During recent years, the biochemical nature of different forms of systemic amyloidosis and variations in pathogenetic mechanisms have been elucidated. The prognosis depends on the biochemical amyloid forms. Nowadays, there are specific treatments available for some systemic amyloidoses. This new development means that exact and safe determination of the type of amyloid deposit in the individual patient is critical. The more or less constant involvement of subcutaneous adipose tissue makes this an attractive site for biopsy for a biochemical characterization of amyloid material. Not only is the tissue a site of significant amyloid deposits—although rarely causing any symptoms—it also contains comparably low levels of other proteins. Furthermore, a
0065-1281/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2006.03.011
ARTICLE IN PRESS 210 surgical biopsy from abdominal subcutaneous fat tissue is simple and without complications. In many cases it is also possible to purify an amyloid fibril protein from a small subcutaneous tissue biopsy (Westermark et al., 1989; Olsen et al., 1998a). We have used abdominal adipose tissue biopsies for determination of the biochemical type of deposits in systemic amyloidosis for many years. Initially, we used double immunodiffusion as a diagnostic tool. Later, we developed a method based on enzyme-linked immunosorbent assay (ELISA) for amyloid diagnostics (Olsen et al., 1999). For the last 3 years, we have used Western blot analysis for clinical assessment of systemic amyloidosis.
Materials and methods Antisera Since commercially available antibodies often fail in analysis of amyloid fibril proteins, we only use in-house raised antisera, produced in rabbits by standard methods. AL-l antiserum (A-132) was raised against fibril protein, purified from the spleen of an individual with systemic amyloidosis. The protein was determined by N-terminal amino acid sequence analysis to be of l-II type (Olsen et al., 1999). AL k antiserum (A-147) was raised against a synthetic peptide corresponding to a Cterminal part of the constant region of k chains (Olsen et al., 1999). The protein AA antiserum (A126) was also raised against a synthetic peptide corresponding to residues 24–34 of human protein AA (Olsen et al., 1999). Transthyretin amyloid was detected by an antiserum (A1898) raised against an in vitro expressed protein corresponding to residues 50–127 of the human protein (Bergstro ¨m et al., 2004). Antisera against apolipoprotein A-IV (A82/ 01) and apolipoprotein A-I (A-159) were also raised in rabbits against synthetic peptides (Bergstro ¨m et al., 2004; Mucchiano et al., 2001).
P. Westermark et al. specimens are taken from the adipose tissue, squashed between two microscope slides, dried for 5 min and defatted in acetone for 10 min. After evaporation, the slides are stained in Congo red B (Puchtler et al., 1962) solution, diluted 1:10 with Congo red A solution, for 10–20 min. This dilution is important since the specimens otherwise tend to become over-stained with a consequent risk of over-diagnosing amyloidosis (Westermark et al., 1999). The slides are then examined directly for the presence of amyloid deposits, without mounting. The remaining part of the biopsy is defatted overnight in several changes of acetone and airdried. Aliquots are put directly into sample buffer containing 3% sodium dodecyl sulfate (SDS) and 0.02 M dithiothreitol, heated to 100 1C for 30 min and incubated at room temperature overnight. SDS–polyacrylamide gel electrophoresis (PAGE) is performed in a standard Tris–tricine buffer system (Scha ¨gger and von Jagow, 1987) and Western blotting is performed as described by Bergstro ¨m et al. (2004) using an enhanced chemiluminescence system (Amersham Biosciences, Uppsala, Sweden). In each analysis, control amyloid fibrilar materials of known AA, Al, Ak and ATTR are included. The primary antisera are diluted 1:2000, except antiATTR which is diluted 1:10,000.
Results Since we developed the present method, 35 abdominal fat biopsies with amyloid deposits have been analyzed in our laboratory. Of these, 32 have been characterized successfully as detailed in Table 1. AL- and ATTR-amyloid are the commonest types. Whether or not the results are due to the occurrence of amyloid fibrils of two biochemical types is not yet known. A band with mobility of a complete immunoglobulin light chain was often obtained with both antisera against AL-proteins. Such bands were regarded as contaminants from
Method
Table 1. Systemic amyloid forms typed by Western blot analysis of subcutaneous fat biopsies 2002–2004
Subcutaneous fat biopsies (2–20 mm in diameter) are sent to our laboratory in 0.15 M NaCl by ordinary mail. The biopsies are rinsed overnight in 0.15 M NaCl–0.02% sodium azide, followed by rinsing with 0.88% ammonium chloride for hemolysis. This step is important for analysis of purified amyloid fibril proteins, because without it a high background of hemoglobin A and B chains becomes problematic. After washing in distilled water, small
Amyloid type
No. of patients
AL-k AL-l Transthyretin AA AApoA-I AApoA-IV Type not determined
2 12 14 4 0 0 3
ARTICLE IN PRESS Subcutis in systemic amyloidosis
211 cases we obtained a reaction with two of the antisera (AA and ATTR), one with strong bands and the other with weak bands. Biopsies were sent to the laboratory at ambient temperature, without cooling. One advantage of abdominal fat biopsies is their relative robustness and resistance to autolysis and no problems with the quality of the material were noted, even in biopsies which had been in the mail over a weekend.
Discussion
Figure 1. Western blot analysis with antiserum against amyloid protein derived from l type of immunoglobulin light chain. Lanes 2 and 3 contain extracts of fat biopsies from patients with systemic amyloidosis of unknown type. Lane 1 contains control material with amyloid of known AL-l type.
Figure 2. Western blot analysis with antiserum against amyloid protein derived from transthyretin. Lanes 1 and 3 contain extracts of fat biopsies from patients with systemic amyloidosis of unknown type. Lane 2 contains control material with known transthyretin-derived amyloid.
plasma and ignored in the interpretation of the immunoblotting results, which were only based on low molecular protein bands (o20 kDa) (Figs. 1 and 2). In three biopsies, no definite amyloid protein band was detected by Western blot. In these cases, the amount of amyloid was very small, with discrete patches or streaks only in some fat tissue fragments. Usually, the result was clear, but in two
The high proportion of individuals with amyloidosis of TTR-origin may seem surprising, but several biopsies were received from a region in Sweden with a high prevalence of familial amyloidotic polyneuropathy (Suhr et al., 2003). The low proportion of patients with AA-amyloidosis is notable. Most likely, biopsies from this category of individuals are under-represented, probably due to clear diagnosis on clinical grounds since AA-amyloidosis is not a rare condition in Sweden (Lo ¨fberg et al., 1987). One of the difficulties in the development of the amyloid typing method was to obtain antisera which specifically recognized the amyloid fibril proteins. There was no problem with protein AA and it was possible to use several of the tested antisera. The reason that we chose an anti-peptide antibody was that this gave no background labeling and was also useful in immunohistochemistry. The commercial anti-TTR antibody that we tested gave good, specific labeling of TTR monomer and dimer bands, but failed to react with TTR fragments, which constitute most of the amyloid fibrils in many cases of Swedish V30M ATTR-amyloidosis and always in wild-type ATTR disease (senile systemic amyloidosis) (Felding et al., 1985; Westermark et al., 1987). On the other hand, the antiserum against TTR50-127 labeled both full-length and fragmented forms of TTR (Bergstro ¨m et al., in preparation). The antisera against AApoA-I and AApoA-IV amyloid proteins were raised against synthetic peptides, since commercial antisera either did not work (apoA-I) or were not available (apoA-IV). These antisera have been shown to recognize the respective amyloid proteins (Mucchiano et al., 2001; Bergstro ¨m et al., 2004). Finding antisera against AL-proteins was a problem. It is accepted that commercial antisera against immunoglobulin light chains are not reliable for detection of AL-amyloid in immunohistochemistry, including immunofluorescence. Such antisera
ARTICLE IN PRESS 212 are raised against the full-length protein. The amyloid protein is usually an N-terminal fragment of an immunoglobulin light chain, and is most usually unfolded. It may therefore contain exposed epitopes that are not recognized by commercial antisera. Antisera against well-characterized proteins purified from amyloid often exhibit remarkable cross-reactivity, but one antiserum, A-132, was shown to have a high specificity for AL-protein of l type. It recognized all l subtypes tested but not amyloid of AL-k type. The antiserum used for recognition of AL-k protein was raised against a short synthetic peptide corresponding to a segment of k constant region. Although most of the ALamyloid proteins consist of N-terminal fragments of monoclonal light chains, fragments of constant region may be found (Eulitz and Linke, 1985). We demonstrated earlier that small fragments of the Cterminal part of the constant region are always present in amyloid deposits of k type (Olsen et al., 1998b) and the same is true for AL-amyloid of l type (Enqvist et al., in preparation). Our studies have shown that these small fragments can be used for determination of the amyloid type (Olsen et al., 1999). However, it has been difficult to raise a specific antiserum against l constant region, which is effective in the analyses. Several methods for the determination of the type of amyloidosis have been described. These include immunohistochemistry at the light (Linke et al., 1995) and electron microscope level (Arbustini et al., 2002), ELISA (Olsen et al., 1999) and biochemical analysis with Edman degradation and mass spectrometry (Murphy et al., 2001). All these methods work in the right hands. The present method has the advantages that it is technically simple and fast. However, the availability of appropriate specific antibodies is a prerequisite.
Acknowledgments Supported by the Swedish Research Council (Project No. 5941), FAMY, FAMY-Norrbotten and the Amyl Foundation.
References Arbustini E, Verga L, Concardi M, Palladini G, Obici L, Merlini G. Electron and immuno-electron microscopy of abdominal fat identifies and characterizes amyloid fibrils in suspected cardiac amyloidosis. Amyloid 2002;9:108–14. Bergstro ¨m J, Murphy CL, Weiss DT, Solomon A, Sletten K, Hellman U, et al. Two different types of amyloid
P. Westermark et al. deposits – apolipoprotein A-IV and transthyretin – in a patient with systemic amyloidosis. Lab Invest 2004; 84:981–8. Duston MA, Skinner M, Shirahama T, Cohen AS. Diagnosis of amyloidosis by abdominal fat aspiration. Analysis of four years’ experience. Am J Med 1987;82:412–4. Eulitz M, Linke R. Amyloid fibrils derived from V-region together with C-region fragments from a lII-immunoglobulin light chain (HAR). Biol Chem H-S 1985;366: 907–15. Felding P, Fex G, Westermark P, Olofsson B- O, Pitka ¨nen P, Benson L. Prealbumin in Swedish patients with senile systemic amyloidosis and familial amyloidotic polyneuropathy. Scand J Immunol 1985;21:133–40. Gertz MA, Li CY, Shirahama T, Kyle RA. Utility of subcutaneous fat aspiration for the diagnosis of systemic amyloidosis (immunoglobulin light chain). Arch Intern Med 1988;148:929–33. Guy CD, Jones CK. Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls. Diagn Cytopathol 2001;24:181–5. Libbey CA, Skinner M, Cohen AS. Use of abdominal fat tissue aspirate in the diagnosis of systemic amyloidosis. Arch Int Med 1983;143:1549–52. Linke RP, Ga ¨rtner HV, Michels H. High-sensitivity diagnosis of AA amyloidosis using Congo red and immunohistochemistry detects missed amyloid deposits. J Histochem Cytochem 1995;43:863–9. Lo ¨fberg H, Grubb A, Thysell H, Nilsson E, Kjellander B, Moller C, et al. The prevalence of renal amyloidosis of the AA-type in a series of 1158 consecutive autopsies. Acta Pathol Microbiol Immunol Scand [A] 1987;95: 297–302. Manganaro M, Bruno M, Torchio B, Pellerito R, Gabella P, Linari F. Abdominal fat aspiration for diagnosis of amyloidosis. Nephron 1992;60:490–1. Maruyama K, Ikeda S, Yanagisawa N, Nakazato M. Diagnostic value of abdominal fat tissue aspirate in familial amyloidotic polyneuropathy. J Neurol Sci 1987;81:11–8. Masouye´ I. Diagnostic screening of systemic amyloidosis by abdominal fat aspiration: an analysis of 100 cases. Am J Dermatopathol 1997;19:41–5. Mucchiano GI, Ha ¨ggqvist B, Sletten K, Westermark P. Apolipoprotein A-1-derived amyloid in atherosclerotic plaques of the human aorta. J Pathol 2001;193: 270–5. Murphy CL, Eulitz M, Hrncic R, Sletten K, Westermark P, Williams T, et al. Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens. Am J Clin Pathol 2001;116:135–42. Olsen KE, Sletten K, Westermark P. Extended analysis of AL-amyloid protein from abdominal wall subcutaneous fat biopsy: kappa IV immunoglobulin light chain. Biochem Biophys Res Commun 1998a;245:713–6. Olsen KE, Sletten K, Westermark P. Fragments of the constant region of immunoglobulin light chain are constituents of AL-amyloid proteins. Biochem Biophys Res Commun 1998b;251:642–7.
ARTICLE IN PRESS Subcutis in systemic amyloidosis Olsen KE, Sletten K, Westermark P. The use of subcutaneous fat tissue for amyloid typing by enzyme-linked immunosorbent assay. Am J Clin Pathol 1999;111: 355–62. Orfila C, Giraud P, Modesto A, Suc J-M. Abdominal fat tissue aspirate in human amyloidosis: light, electron, and immunofluorescence microscopic studies. Hum Pathol 1986;17:366–9. Puchtler H, Sweat F, Levine M. On the binding of Congo red by amyloid. J Histochem Cytochem 1962;10: 355–64. Scha ¨gger H, von Jagow G. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166:368–79. ¨ ber die amyloide Entartung der Haut. Frankf Schilder P. U Z Pathol 1909;3:782–94. Suhr OB, Svendsen IH, Andersson R, Danielsson A, Holmgren G, Ranlo ¨v PJ. Hereditary transthyretin amyloidosis from a Scandinavian perspective. J Intern Med 2003;254:225–35.
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