/oirrrid of Iriterncrl Medicine 1992 : 232, 5 19-52 1
Amyloid P component and the diagnosis of amyloidosis M. B. PEPYS From the Kogal Postgraduate Medical School. Hammersmith Hospital. London. U K
Introduction Amyloidosis can affect any part of the body, producing protean manifestations, and it can never be diagnosed on purely clinical grounds. Histologic examination is the standard method, but the recent development of isotope-labelled serum amyloid P component as a specific tracer for amyloid in vivo enables the diagnosis to be made directly in vivo and yields substantially more information than can be obtained from biopsies [l].
The function(s) of SAP may be related to its binding reactions with DNA, chromatin, glycosaminoglycans, fibronectin. and C4-binding protein [2, 41. SAP is also a normal constituent of the glomerulat basement membrane and elastic fiber microfibrils [2].
Amyloid P component
Biopsy
Amyloid deposits consist of amyloid fibrils with their associated glycosaminoglycans. but they also always contain a non-fibrillar glycoprotein, amyloid P component (AP), which comprises up to 1 5 % of the deposit. AP is identical to and derived from the normal circulating plasma protein, serum amyloid P component (SAP), which is a member of the pentraxin family of proteins, the other member of which in humans is C-reactive protein [21. AP is deposited in amyloid because SAP is a calcium-dependent ligandbinding protein that recognizes and binds to structure(s) (probably the glycosaminoglycans) present on all types of amyloid fibrils. It is not known whether SAP or AP plays any role in the pathogenesis of amyloid fibril deposition or its persistence. No deficiency or polymorphism of SAP has been described, and there is a single copy of the gene in humans on chromosome 1. Homologous proteins are present in all vertebrates. The single biantennary Nlinked oligosaccharide present on each of the 10 identical polypeptide subunits of SAP (25462 Da each) is the most invariant glycan of any known mammalian glycoprotein. Circulating SAP is produced by hepatocytes, and its plasma concentration is tightly regulated (women : mean, 24 mg-' ; SD, 8 ; range, 8-55 [n = 2741; men: mean, 32 mg-I ; SD, 7 ; range. 12-50 [ti = 2261). The level remains normal even during massive deposition of SAP into amyloid 131.
Amyloid may be an incidental finding on biopsy of the kidneys, liver, heart, bowel, peripheral nerve, lymph node, thyroid, or bone marrow. When amyloidosis is suspected clinically, biopsy of rectum or subcutaneous fat is the least invasive. Amyloid is present in these sites in more than 90% of cases of systemic AA (secondary amyloidosis) or AL (primary amyloidosis). Alternatively, a clinically affected tissue may undergo direct biopsy. Fixation in neutral buffered formalin and wax embedding are satisfactory, although most staining reactions are most intense on cryostat sections of fresh-frozen tissue.
Histochemical diagnosis of amyloid
Congo red and other histochemical stains Many cotton dyes, fluorochromes, and metachromatic stains have been used, but Congo red staining, and its resultant green birefringence when viewed with high-intensity polarized light, is the pathognomonic histochemical test for amyloidosis [S]. The stain is unstable and must be freshly prepared every 2 months or less. Section thickness of 5-10 ~ L Mand inclusion in every staining run of a positive control tissue containing modest amounts of amyloid are critical. Physical and chemical procedures (permanganate, trypsin, autoclaving) that differentially modify the congophilia of different types of amyloid fibrils have been rendered obsolete by the general availability of immunohistochemical reagents. 519
520
M. PEPYS
lniniunohistochenristry Although many amyloid fibril proteins can be identified immunohistochemically, the demonstration of amyloidogenic proteins in tissues does not, on its own, establish the presence of amyloid. Congo red staining and green birefringence are always required, and immunostaining may then enable the amyloid to be classified. Antibodies to serum amyloid A (AA) are commercially available and always stain AA deposits, similarly with anti-p,-microglobulin antisera and haemodialysis-associated amyloid. Only about half of all AL amyloids are stainable with standard antisera to K or R, probably because the light-chain fragment in the fibrils is usually the Nterminal variable domain, which is largely unique for each monoclonal protein. Immunohistochemical staining of transthyretin, /l protein, and prion protein amyloids may require pretreatment of sections with formic acid or alkaline guanidine or deglycosylation.
Electron microscopy Amyloid fibrils cannot always be convincingly identified ultrastructurally, and electron microscopy alone is not sufficient to confirm the diagnosis of amyloidosis.
Problems of histologic diagnosis The tissue sample must be adequate (for example, the inclusion of submucosal vessels in a rectal biopsy specimen), and failure to find amyloid does not exclude the diagnosis. The unavoidable problem of sampling error means that biopsy cannot reveal the extent or distribution of amyloid. Experience with Congo red staining is required if clinically important false-negative and false-positive results are to be avoided. Immunohistochemical staining requires positive and negative controls, including demonstration of specificity of staining by absorption of positive antisera with isolated pure antigens.
Non-histologic investigations Two-dimensional echocardiography showing small, concentrically hypertrophied ventricles, generally impaired contraction, dilated atria, homogeneously echogenic valves, and ‘sparkling ’ echodensity of ventricular walls can be diagnostic of cardiac
amyloidosis. However, some of the signs may be absent despite histologically confirmed involvement. Imaging after injection of isotope-labelled calciumseeking tracers has poor sensitivity and specificity and is of no clinical use.
Serum amyloid P component as a specific tracer in amyloidosis The universal presence in amyloid deposits of AP, derived from circulating SAP, suggested the use of radioisotope-labelled SAP as a diagnostic tracer in amyloidosis. No localization or retention of labelled SAP occurs in healthy subjects or in patients with diseases other than amyloidosis. Radioiodinated SAP has a short half-life (24 h) in the plasma and is rapidly catabolized : there is complete excretion of the iodinated breakdown products in the urine [2]. However, in patients with amyloidosis. the tracer rapidly and specifically localizes to the deposits, in proportion to the quantity of amyloid present, and persists there without breakdown or modification. For clinical purposes, highly purified SAP is isolated from the plasma of single accredited donors and is oxidatively iodinated under conditions that preserve its function intact. The medium-energy, short halflife, pure gamma emitter lZ3T is used for scintigraphic imaging, and the long half-life isotope is used for metabolic studies. The doses of radioactivity administered (less than 4 mSv) are well within accepted safety limits. More than 500 studies have been completed without any adverse effects in more than 250 patients and 1 2 0 controls. Important observations regarding amyloid (which have been made for the first time in vivo) include the following : the different distribution of amyloid in different forms of the disease; amyloid in anatomic sites not available for biopsy (adrenals, spleen) : major systemic deposits in forms of amyloid previously thought to be organ-limited: a poor correlation between the quantity of amyloid present in a given organ and the level of organ dysfunction: a nonhomogeneous distribution of amyloid within individual organs ; and evidence for rapid progression and sometimes regression of amyloid deposits with different rates in different organs. Major regression of amyloidosis, when it has been possible to reduce or eliminate the supply of fibril precursor protein, is very encouraging. Labelled SAP makes a valuable contribution to the diagnosis and management of patients with systemic amyloidosis, and although it
D I A G N O S I S W I T H AMYLOID P C O M P O N E N T
is still a highly specialized tool of restricted availability, it is hoped that it will become more accessible and widely used in the near future.
References 1 Hawkins 1".
Lavender JP, Pepys MB. Evaluation of systemic amyloidosis by scintigraphy with '"I-labelled serum amyloid P component. N E r i g l / Med 1 9 9 0 : 3 2 3 : 508-13. 2 Pcpys MB. Baltz ML. Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein. Adv lmrnunol 1 9 8 3 : 34: 14 1-2 12.
521
3 Hawkins PN. Wootton R. Pepys MB. Metabolic studies of radioiodinated serum amyloid P component in normal subjects and patients with systemic amyloidosis. J Clin Invest 1 9 9 0 : 86: 1862-9. 4 Butler PJG. Tennent GA. Pepys MB. Pentraxin-chromatin interactions : serum amyloid P component specifically displaces HI -type histones and solubilizes native long chromatin. J E x p Med 1 9 9 0 ; 172: 13-8. 5 Puchtler H. Sweat F. Levine M. On the binding of Congo red by amyloid. I Histochem Cytochern 1962: 10: 355-64. Received 1 0 June 1 9 9 2 ; accepted 23 Junc 1992.
Correspondence; Mayo Clinic. 200 First Street SW. Rochester, Minnesota 55905, USA.
Amyloid P component and the diagnosis of amyloidosis M. B. PEPYS From the Kogal Postgraduate Medical School. Hammersmith Hospital. London. U K
Introduction Amyloidosis can affect any part of the body, producing protean manifestations, and it can never be diagnosed on purely clinical grounds. Histologic examination is the standard method, but the recent development of isotope-labelled serum amyloid P component as a specific tracer for amyloid in vivo enables the diagnosis to be made directly in vivo and yields substantially more information than can be obtained from biopsies [l].
The function(s) of SAP may be related to its binding reactions with DNA, chromatin, glycosaminoglycans, fibronectin. and C4-binding protein [2, 41. SAP is also a normal constituent of the glomerulat basement membrane and elastic fiber microfibrils [2].
Amyloid P component
Biopsy
Amyloid deposits consist of amyloid fibrils with their associated glycosaminoglycans. but they also always contain a non-fibrillar glycoprotein, amyloid P component (AP), which comprises up to 1 5 % of the deposit. AP is identical to and derived from the normal circulating plasma protein, serum amyloid P component (SAP), which is a member of the pentraxin family of proteins, the other member of which in humans is C-reactive protein [21. AP is deposited in amyloid because SAP is a calcium-dependent ligandbinding protein that recognizes and binds to structure(s) (probably the glycosaminoglycans) present on all types of amyloid fibrils. It is not known whether SAP or AP plays any role in the pathogenesis of amyloid fibril deposition or its persistence. No deficiency or polymorphism of SAP has been described, and there is a single copy of the gene in humans on chromosome 1. Homologous proteins are present in all vertebrates. The single biantennary Nlinked oligosaccharide present on each of the 10 identical polypeptide subunits of SAP (25462 Da each) is the most invariant glycan of any known mammalian glycoprotein. Circulating SAP is produced by hepatocytes, and its plasma concentration is tightly regulated (women : mean, 24 mg-' ; SD, 8 ; range, 8-55 [n = 2741; men: mean, 32 mg-I ; SD, 7 ; range. 12-50 [ti = 2261). The level remains normal even during massive deposition of SAP into amyloid 131.
Amyloid may be an incidental finding on biopsy of the kidneys, liver, heart, bowel, peripheral nerve, lymph node, thyroid, or bone marrow. When amyloidosis is suspected clinically, biopsy of rectum or subcutaneous fat is the least invasive. Amyloid is present in these sites in more than 90% of cases of systemic AA (secondary amyloidosis) or AL (primary amyloidosis). Alternatively, a clinically affected tissue may undergo direct biopsy. Fixation in neutral buffered formalin and wax embedding are satisfactory, although most staining reactions are most intense on cryostat sections of fresh-frozen tissue.
Histochemical diagnosis of amyloid
Congo red and other histochemical stains Many cotton dyes, fluorochromes, and metachromatic stains have been used, but Congo red staining, and its resultant green birefringence when viewed with high-intensity polarized light, is the pathognomonic histochemical test for amyloidosis [S]. The stain is unstable and must be freshly prepared every 2 months or less. Section thickness of 5-10 ~ L Mand inclusion in every staining run of a positive control tissue containing modest amounts of amyloid are critical. Physical and chemical procedures (permanganate, trypsin, autoclaving) that differentially modify the congophilia of different types of amyloid fibrils have been rendered obsolete by the general availability of immunohistochemical reagents. 519
520
M. PEPYS
lniniunohistochenristry Although many amyloid fibril proteins can be identified immunohistochemically, the demonstration of amyloidogenic proteins in tissues does not, on its own, establish the presence of amyloid. Congo red staining and green birefringence are always required, and immunostaining may then enable the amyloid to be classified. Antibodies to serum amyloid A (AA) are commercially available and always stain AA deposits, similarly with anti-p,-microglobulin antisera and haemodialysis-associated amyloid. Only about half of all AL amyloids are stainable with standard antisera to K or R, probably because the light-chain fragment in the fibrils is usually the Nterminal variable domain, which is largely unique for each monoclonal protein. Immunohistochemical staining of transthyretin, /l protein, and prion protein amyloids may require pretreatment of sections with formic acid or alkaline guanidine or deglycosylation.
Electron microscopy Amyloid fibrils cannot always be convincingly identified ultrastructurally, and electron microscopy alone is not sufficient to confirm the diagnosis of amyloidosis.
Problems of histologic diagnosis The tissue sample must be adequate (for example, the inclusion of submucosal vessels in a rectal biopsy specimen), and failure to find amyloid does not exclude the diagnosis. The unavoidable problem of sampling error means that biopsy cannot reveal the extent or distribution of amyloid. Experience with Congo red staining is required if clinically important false-negative and false-positive results are to be avoided. Immunohistochemical staining requires positive and negative controls, including demonstration of specificity of staining by absorption of positive antisera with isolated pure antigens.
Non-histologic investigations Two-dimensional echocardiography showing small, concentrically hypertrophied ventricles, generally impaired contraction, dilated atria, homogeneously echogenic valves, and ‘sparkling ’ echodensity of ventricular walls can be diagnostic of cardiac
amyloidosis. However, some of the signs may be absent despite histologically confirmed involvement. Imaging after injection of isotope-labelled calciumseeking tracers has poor sensitivity and specificity and is of no clinical use.
Serum amyloid P component as a specific tracer in amyloidosis The universal presence in amyloid deposits of AP, derived from circulating SAP, suggested the use of radioisotope-labelled SAP as a diagnostic tracer in amyloidosis. No localization or retention of labelled SAP occurs in healthy subjects or in patients with diseases other than amyloidosis. Radioiodinated SAP has a short half-life (24 h) in the plasma and is rapidly catabolized : there is complete excretion of the iodinated breakdown products in the urine [2]. However, in patients with amyloidosis. the tracer rapidly and specifically localizes to the deposits, in proportion to the quantity of amyloid present, and persists there without breakdown or modification. For clinical purposes, highly purified SAP is isolated from the plasma of single accredited donors and is oxidatively iodinated under conditions that preserve its function intact. The medium-energy, short halflife, pure gamma emitter lZ3T is used for scintigraphic imaging, and the long half-life isotope is used for metabolic studies. The doses of radioactivity administered (less than 4 mSv) are well within accepted safety limits. More than 500 studies have been completed without any adverse effects in more than 250 patients and 1 2 0 controls. Important observations regarding amyloid (which have been made for the first time in vivo) include the following : the different distribution of amyloid in different forms of the disease; amyloid in anatomic sites not available for biopsy (adrenals, spleen) : major systemic deposits in forms of amyloid previously thought to be organ-limited: a poor correlation between the quantity of amyloid present in a given organ and the level of organ dysfunction: a nonhomogeneous distribution of amyloid within individual organs ; and evidence for rapid progression and sometimes regression of amyloid deposits with different rates in different organs. Major regression of amyloidosis, when it has been possible to reduce or eliminate the supply of fibril precursor protein, is very encouraging. Labelled SAP makes a valuable contribution to the diagnosis and management of patients with systemic amyloidosis, and although it
D I A G N O S I S W I T H AMYLOID P C O M P O N E N T
is still a highly specialized tool of restricted availability, it is hoped that it will become more accessible and widely used in the near future.
References 1 Hawkins 1".
Lavender JP, Pepys MB. Evaluation of systemic amyloidosis by scintigraphy with '"I-labelled serum amyloid P component. N E r i g l / Med 1 9 9 0 : 3 2 3 : 508-13. 2 Pcpys MB. Baltz ML. Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein. Adv lmrnunol 1 9 8 3 : 34: 14 1-2 12.
521
3 Hawkins PN. Wootton R. Pepys MB. Metabolic studies of radioiodinated serum amyloid P component in normal subjects and patients with systemic amyloidosis. J Clin Invest 1 9 9 0 : 86: 1862-9. 4 Butler PJG. Tennent GA. Pepys MB. Pentraxin-chromatin interactions : serum amyloid P component specifically displaces HI -type histones and solubilizes native long chromatin. J E x p Med 1 9 9 0 ; 172: 13-8. 5 Puchtler H. Sweat F. Levine M. On the binding of Congo red by amyloid. I Histochem Cytochern 1962: 10: 355-64. Received 1 0 June 1 9 9 2 ; accepted 23 Junc 1992.
Correspondence; Mayo Clinic. 200 First Street SW. Rochester, Minnesota 55905, USA.
