How to diagnose amyloidosis

Authors

  • P. Mollee,

    Corresponding author
    1. Haematology Department, Pathology Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia
    2. School of Medicine, University of Queensland, Brisbane, Queensland, Australia
    • Correspondence

      Peter Mollee, Haematology Department, Pathology Queensland, Princess Alexandra Hospital, Ipswich Road, Brisbane, Qld 4102, Australia.

      Email: peter_mollee@health.qld.gov.au

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  • P. Renaut,

    1. Anatomical Pathology Department, Pathology Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia
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  • D. Gottlieb,

    1. Haematology Department, Westmead Hospital, Sydney, New South Wales, Australia
    2. Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
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  • H. Goodman

    1. Haematology Department, Waikato Hospital, Hamilton, New Zealand
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  • Funding: None.
  • Conflict of interest: None.

Abstract

Amyloidosis is a rare but devastating condition caused by deposition of misfolded proteins as aggregates in the extracellular tissues of the body, leading to impairment of organ function. High clinical suspicion is required to facilitate early diagnosis. Correct identification of the causal amyloid protein is absolutely crucial for clinical management in order to avoid misdiagnosis and inappropriate, potentially harmful treatment, to assess prognosis, and to offer genetic counselling if relevant. This review summarises the current evidence on which the diagnosis and subtyping of amyloidosis is based, outlines the limitations of various diagnostic techniques, particularly in an Australian and New Zealand context, and discusses optimal strategies for the diagnostic approach to these patients. Recommendations are provided for when particularly to suspect amyloidosis, what investigations are required, as well as an approach to accurate subtyping of amyloidosis.

Introduction

For the majority of physicians, amyloidosis is a mysterious disease, often only considered following an unexpected pathology report. Over the years, variations in classification systems have not helped the understanding of the disease. This is a pity because fundamentally amyloidosis is a simple disease characterised by tissue deposition of a protein able to assume an insoluble beta-pleated sheet structure. This tissue deposition ultimately interferes with normal organ function. The physician faced with a patient with a new diagnosis of amyloidosis initially must ask himself or herself only three important questions: what is the protein that is causing the amyloidosis, where is it being produced, and finally, in what tissues of the body is it being deposited? All therapeutic decisions will logically follow.

Approximately 25 proteins currently are recognised to cause amyloidosis and the amyloidogenic protein is the basis for the current classification (Table 1). Each type of amyloid has the prefix ‘A’, for amyloid, followed by an abbreviation derived from the name of the protein; thus, AL designates amyloid derived from immunoglobulin light chain, ATTR is amyloid derived from transthyretin (TTR), AFib indicates amyloid derived from fibrinogen, etc. In amyloidosis, these normally soluble proteins misfold and aggregate to form protofilaments and fibrils by virtue of a common cross beta-pleated sheet structure. The fibrils then co-deposit in the extracellular space with serum amyloid P protein (SAP) and other components, such as glycosaminoglycans, to form the insoluble amyloid deposits.

Table 1. Amyloid subtypes and their associated clinical phenotypes
Amyloid typeOther namesProteinMechanismOrgans involvedAssociated disease/population
KidneyHeartLiverGITNervesSoft tissueOther
  1. ++++, 50–100%; +++, 25–50%; ++, 10–25; +, <10%; -, organ involvement not reported or uncommon. †Symptomatic macroglossia and skeletal muscle involvement are highly suggestive of the AL subtype. ‡Factor X deficiency (or, rarely, other factors) has only been reported in AL subtype. §Amyloid deposits in bone cysts and joint synovium. ¶Facial paresis, also often associated with a sensory peripheral neuropathy. GIT, gastrointestinal tract; NA, not applicable.
AL[1]Primary (no longer favoured terminology)Monoclonal Ig light chainsAcquired, abnormal, amyloid-forming protein+++++++++++++++++Acquired factor X deficiency ++Plasma cell dyscrasia/older people
AA[2]Secondary/reactive (no longer favoured terminology)Serum amyloid ANormal protein at supra-normal concentration+++++++NAAny cause of systemic inflammation
AB2M[3]DRA (dialysis related amyloidosis)Beta-2 microglobulinNormal protein at supra-normal concentration++++§NADialysis
ATTRwt (wild type)[4]Senile systemic (cardiac) amyloidosisTransthyretin (wild type)Normal protein, normal concentration, prolonged exposure+++++Carpal tunnel syndrome +++Older age (>80), usually male
ATTRm (mutated)[5]FAP or FAC (familial amyloid polyneuropathy/cardiomyopathy)Transthyretin (mutated)Hereditary, mutant protein++++++++

Leptomeningeal +

Vitreous opacities +

Different population groups depending on mutation
AFib[6]NAFibrinogen A-alpha chainHereditary, mutant protein+++++NANA
ALys[7]NALysozymeHereditary, mutant protein+++++++++Splenic ruptureNA
AApoA1 and 2[8]NAApolipoproteins 1 and 2Hereditary, mutant protein++++++++++++NANA
ALect2[9]NALeukocyte chemotactic factor 2Hereditary, mutant protein++++NAHispanic Americans
AGel[10]Finnish; Meretoja syndromeGelsolinHereditary, mutant proteinCranial neuropathy ++++Corneal lattice dystrophy ++++NA

Like a group of naughty children, each amyloid-producing protein produces its own form of mischief. Some cause chaos rapidly, others in a slower but nevertheless relentless fashion. Some have predilections for particular organs such as the myocardium, the kidneys or the nerves. It can be difficult to recognise these patterns outside the few centres reviewing large numbers of patients with this rare disease and therefore centralised review if not management of all such patients should be considered. In the meantime, amyloid can be demystified. The aim of this paper is to provide physicians with a framework for the assessment and diagnosis of suspected and proven amyloidosis. Management of amyloidosis is beyond the scope of this article, and readers are referred to recent reviews on this topic.[11, 12]

When to suspect amyloidosis

Amyloidosis can present with a bewildering array of symptoms depending on the organs involved (Fig. 1). Initial symptoms are often non-specific, such as fatigue and weight loss, but as the disease progresses, symptomatology reflects the impairment of the organs involved by the amyloidosis. Certain clinical presentations require a diagnosis of amyloidosis to be considered (Table 2). These include nephrotic range proteinuria, cardiac failure with left ventricular hypertrophy in the absence of hypertension or aortic valve disease, sensorimotor peripheral neuropathy without obvious cause, and hepatomegaly with a normal appearance on ultrasound or computed tomography (CT) imaging. As the most common type of amyloidosis is AL, the combination of an appropriate clinical scenario and an immunoglobulin free light chain (FLC) abnormality (see later discussion) provides a high suspicion of AL amyloidosis necessitating further histological investigation.

Figure 1.

Organ involvement in common subtypes of amyloidosis (++++, 50–100%; +++, 25–50%; ++, 10–25%; +, <10%). Illustration modified from Anatomica, the complete home medical reference[44] and used with permission from Global Book Publishing. AFib, amyloid derived from fibrinogen; AL, amyloid derived from immunoglobulin light chain; ATTR, amyloid derived from transthyretin.

Table 2. More common clinical scenarios where amyloidosis should be suspected
Nephrotic range proteinuria
Cardiac failure with left ventricular hypertrophy in the absence of hypertension or aortic valve disease
Sensorimotor peripheral neuropathy without obvious cause
Hepatomegaly with a normal appearance on ultrasound or computed tomography imaging
Autonomic neuropathy

The amyloidoses are most often systemic that is where the production of the amyloid-forming protein is distant to the amyloid deposits (e.g., monoclonal immunoglobulin production in the bone marrow depositing as amyloid in the heart, or variant fibrinogen produced in the liver depositing as amyloid in the kidney). Localised amyloidosis, amyloid deposits occurring only at the site of amyloid-forming protein production, is another well-recognised entity. Localised amyloidosis has a range of well-recognised presentations, particularly those because of localised AL amyloid that is a non-life-threatening disease with rare progression to systemic AL amyloidosis but frequent local recurrences.[13] Localised amyloidosis results from the local production and deposition of amyloidogeneic proteins, with AL type deposits thought to be produced by foci of low-grade monoclonal B-cells or plasma cells that secrete monoclonal immunoglobulin light chains in the immediate vicinity.[14] These amyloid deposits are commonly located in the airways (including nasopharynx, larynx and bronchi) and lungs, orbit and adnexae, bladder, gastrointestinal tract, lymph nodes, and skin.[15] Localised amyloidosis can and has been reported to occur in almost any organ of the body. It is also seen infiltrating plasmacytomas and, in this situation, is not necessarily indicative of systemic disease. Occasionally localised amyloid can be seen in the skin comprised of insulin around injection sites of insulin-dependent diabetics or of keratin in areas of excoriation or trauma.

Making the diagnosis of amyloidosis

The diagnosis of amyloidosis requires a tissue biopsy. To date, the gold standard test for histological confirmation of amyloid deposits is the Congo red stain used in conjunction with polarised light microscopy. Congo red results in a pale ‘salmon-pink’ staining that shows typical birefringence and dichroism effects when examined under polarised light microscopy (Fig. 2). It is essential that a reliable staining protocol is used to avoid non-specific staining.[16, 17] In some cases, multiple sections may need to be examined as amyloid deposits can be focal and irregularly distributed.[18] In circumstances where there is a high index of suspicion for amyloidosis and Congo red appears to be negative, an alternative dye such as crystal violet may be used but can be non-specific. The fact that SAP, which is a normal plasma glycoprotein, is present in all types of amyloid,[19] has been exploited to aid in the diagnosis of amyloid by immunohistochemistry, but the staining pattern that is obtained with antibodies to SAP can be difficult to interpret. Hence, this test should not be used in isolation for the diagnosis of amyloidosis. Electron microscopy (EM) is often used in conjunction with histology for the diagnosis of amyloidosis, although this is not always necessary. Ultrastructurally, amyloid deposits are composed of haphazardly distributed, non-branching fibrils with a mean diameter of 10 nm (range 8–12 nm) and an electron-lucent core.[20] This feature is characteristic, but not specific, as fibril deposition may be seen as other conditions, such as fibrillary glomerulonephritis, immunotactoid glomerulonephritis, glomerular sclerosis, diabetic fibrillosis, fibronectin glomerulopathy and collagenofibrotic glomerulopathy.[21] These are differentiated according to fibril appearance and diameter, and the findings should be interpreted in conjunction with those of light microscopy, Congo red staining and immunofluorescence or immunohistochemistry.

Figure 2.

(A) Congo red staining demonstrating amyloid deposition in a cardiac biopsy sample (×40); (B) Congo red staining demonstrating that the deposits exhibit apple-green birefringence under polarised light (×40).

Not all monoclonal immunoglobulin deposition in the tissues is due to amyloidosis. In rare cases, monoclonal immunoglobulin can be deposited particularly in the kidneys in a non-amyloid form.[22] These diseases (e.g. light and heavy chain deposition disease) do not have the typical Congo red staining under polarised light, have a tendency towards kappa rather than lambda light chain deposition and in general have more restricted organ involvement than AL amyloid with which they are often confused. Like AL amyloid, however, they are frequently associated with myeloma or other lymphoproliferative diseases.

What to biopsy

For the diagnosis of amyloidosis, biopsy of the clinically involved organ is the most sensitive method and has the advantages of providing larger amounts of tissue for subsequent subtyping and detecting concomitant pathologies. However, such biopsies can be associated with discomfort and the morbidity of bleeding and rarely organ perforation. Thus, if amyloidosis is suspected, a less invasive biopsy may be preferred and can be taken from a distant site such as the abdominal fat, bone marrow, rectum, gingiva or minor salivary glands. Reports from reference centres suggest high sensitivity – for example, amyloid deposits can be detected in the bone marrow trephine in 70% of cases[23] and in fat pad (aspirate or biopsy) and rectal biopsies in over 80%[23, 24] – but in our experience, this is difficult to replicate in more general settings.[25] Amyloid deposits on such screening biopsies are often very small, and there is the disadvantage of limited material for subsequent subtyping that may necessitate the need for further biopsy. Nevertheless, abdominal fat pad aspiration has been used increasingly in recent years, and a description and instructional video of the procedure can be found online.[26] Careful collaboration with the pathology service is required when setting up this procedure. If initial screening biopsies are negative and the clinical suspicion of amyloidosis is high, then screening biopsies may need to be repeated or the clinically affected organ should be biopsied.

Key points related to diagnosis of amyloidosis

  • Early diagnosis is the key to effective management
  • The diagnosis of amyloidosis requires a high index of clinical suspicion, particularly in certain clinical presentations (Table 2)
  • Amyloidosis cannot be diagnosed without biopsy of either an affected organ or an amyloid-containing but clinically silent site
  • Screening biopsy of abdominal fat may be useful to confirm the diagnosis and avoid biopsy of major organs, but sensitivity is only moderate
  • Congo red staining of a biopsy sample remains the gold standard diagnostic test.

Subtyping of amyloidosis: getting the diagnosis right

Once amyloidosis is confirmed, it is of critical importance to identify the subtype accurately, as management differs substantially depending on the nature and source of the amyloid-forming protein ranging from supportive care through to aggressive chemotherapy or organ transplantation. The approach to subtyping has evolved dramatically over the last 30 years, moving from limited, uninformative histology stains through more specific immunohistochemistry supplemented by genetic analyses and, latterly, including direct identification of the amyloid-forming protein in biopsy specimens using tandem mass spectrometry. Every amyloid patient now can – and should – have his/her amyloid protein identified to a high level of confidence. This requires careful consideration of the patient's presentation and phenotype, the presence or absence of associated diseases, and findings of histopathology, genetic testing and direct analysis of fibril proteins.

Importance of clinical phenotype

Systemic amyloidoses may affect any major organ, with the notable exception of the brain parenchyma, and the clinical phenotypes are therefore protean. Patients must therefore be assessed for organ involvement as there are some broad subtype–phenotype associations that help inform diagnosis of amyloid subtype and subsequent treatment choices. The clinical and laboratory/imaging features of involvement in various organs are summarised in Table 3. Work-up must include clinical assessment considering all the features in Table 3, in particular investigations of renal function (serum creatinine, 24-h proteinuria), heart (brain natriuretic peptide (BNP), troponin, electrocardiogram (ECG) and echocardiography) and liver (liver function tests and ultrasound scan for span if clinically uncertain).

Table 3. Investigating organ involvement in amyloidosis
OrganClinical findingUseful investigations and findings that suggest involvementISA consensus definition for organ involvement in AL amyloidosisa[27]
  1. aOrgans are considered involved if amyloid present in a biopsy of that organ (excluding blood vessels as the sole site of amyloid deposition), or proven in another site and meeting the clinical criteria above. ALP, alkaline phosphatase; CT, computed tomography; GGT, gamma glutamyl transpeptidase; GIT, gastrointestinal; ISA, International Society of Amyloidosis; NT-ProBNP, N-terminal prohormone of brain natriuretic peptide; ULN, upper limit of normal; USS, ultrasound scan.
KidneyOedema

Proteinuria

Renal failure

No particular imaging features

24-h urine protein >500 mg/day, predominantly albumin
Heart

Breathlessness on exertion

Heart failure

Arrhythmias less common

Raised NT-ProBNP and/or troponin ECG often characteristic in AL (low-voltage, poor R-wave progression)

Echocardiograph: ventricular and valve thickening, biatrial enlargement, diastolic physiology

MRI (late gadolinium enhancement characteristic)

NT-proBNP ≥332 ng/L, in the absence of renal failure or atrial fibrillation

Mean left ventricular wall thickness >12 mm, no other cardiac cause

LiverHepatomegaly

Abnormal liver function, usually ALP/GGT

USS for size (if clinically uncertain)

No other particular imaging features

Span >15 cm in the absence of heart failure

ALP >1.5 × ULN

GIT

Symptoms can be difficult to distinguish from autonomic neuropathy

Gastrointestinal bleeding

No particular imaging features

Biopsy, but do only if clinically important to establish involvement

Direct biopsy verification with symptoms
Nerve

Peripheral neuropathy (distal, symmetrical, sensory neuropathy; motor neuropathy uncommon in AL)

Autonomic neuropathy (early satiety, irregular bowel habit, erectile dysfunction, postural hypotension)

Formal nerve conduction studies not usually helpful.

Nerve biopsy, but do only if clinically important to establish involvement.

Clinical diagnosis
Soft tissue

Macroglossia

Arthropathy

Claudication (vascular amyloid)

Myopathy (pseudohypertrophy)

Biopsy, but do only if clinically important to establish involvement 
LungBreathlessness, cough

CT, interstitial pattern (in absence of pulmonary oedema)

Biopsy, but do only if clinically important to establish involvement

Interstitial radiographic pattern in absence of pulmonary oedema

Direct biopsy verification with symptoms

For assessment of heart involvement by amyloid, the cardiac biomarkers are important. Similar to its role in heart failure, a N-terminal prohormone of BNP <332 ng/L effectively excludes important cardiac amyloid.[27] Echocardiography remains an important screening tool, but it should be noted that the classic ‘speckled appearance’ in the myocardium is a late feature and its absence by no means excludes significant cardiac involvement. Typical features are a thick-walled left ventricle because of amyloid infiltration, a preserved ejection fraction, biatrial enlargement and restrictive filling patterns on Doppler studies; however, no echocardiographic appearance is specific for amyloid heart disease. Cardiac magnetic resonance imaging (MRI) is a useful investigation in select cases to confirm the presence and potential severity of cardiac involvement in AL and ATTR,[28] although significant renal dysfunction is a relative contraindication because of the rare risk of nephrogenic systemic sclerosis. Late gadolinium enhancement is a characteristic and relatively specific finding.[29]

The relationship between clinical phenotype – that is, the patient and their organ involvement – and amyloid subtype is summarised in Table 1 and discussed briefly as follows.

  • AL amyloidosis is more common in older patients as the incidence of monoclonal gammopathies, from which the amyloid-forming monoclonal immunoglobulin light chains are derived, increases with age. It can affect one or many organ systems, the multitude or pattern of which often makes other amyloidoses unlikely. Periorbital and other bruising perhaps because of the fragility of amyloid-affected vessels are more common in AL amyloidosis than other subtypes; symptomatic macroglossia, skeletal muscle involvement and coagulation factor X deficiency, and subtle thickening of the tissues of the lower face are highly suggestive of AL amyloidosis but are each seen in only a small minority.[1]
  • AA amyloidosis primarily affects the kidney with later involvement of the liver and sometimes the gastrointestinal tract. Symptomatic cardiac and nerve involvement are rare.[2]
  • ATTR amyloidosis primarily affects the heart and peripheral and/or autonomic nervous system. The unmutated TTR molecule causes senile systemic amyloidosis (also known as senile cardiac amyloidosis) that has a cardiac-dominant presentation in the very elderly.[4] There are at least 100 recognised mutations of the TTR molecule that increase its amyloidogenicity, understandably producing some phenotypic heterogeneity.[5] The commonest mutation worldwide is the Ile122 that is found in ∼4% of west Africans (including African Americans) and causes slow-onset cardiac amyloidosis in the 7th and 8th decades.[30] Many mutations, including Met30 and Ala60, are seen at low frequency in the Australasian population.
  • AFib amyloidosis is one of the more common hereditary amyloidoses in Australasia.[31] It is a renal-dominant amyloidosis with characteristic presentation of slowly progressive nephrotic syndrome and renal failure in the 6th–7th decades.[6]
  • Many other hereditary amyloidoses are recognised and are summarised briefly in Table 1. ALect2 amyloidosis was only described recently,[9] and it is likely that novel amyloid-forming proteins will be identified as direct protein identification techniques are implemented more widely.

A small number of centres internationally, but not in Australia or New Zealand, has access to targeted scintigraphy of amyloid deposits using I-131-labelled SAP.[32] This allows identification of amyloid in larger viscera such as the kidneys, liver and spleen, as well as bones. Resolution is generally low, but the distribution of amyloid may provide a clue to amyloid type. While SAP scintigraphy is a very useful ancillary technique, the majority of diagnostic and monitoring information required for patient management can be gained from other investigations. The heart is also not imaged by this technique, but reports over many years suggest that technetium scintigraphy (e.g. technetium(99mTc)3,3-diphosphono-1,2- propanedicarboxylic acid) may allow identification of cardiac involvement.[33]

Searching for associated diseases

As one considers the patient and their phenotype, so must a search begin for any potentially associated diseases such as a plasma cell dyscrasia or chronic inflammation. However, it must be appreciated that association is not evidence of causality. For example, a monoclonal gammopathy of uncertain significance (MGUS, or benign paraprotein) is found in ∼3% of Australian adults in their 50s, rising to 9% in their 80s,[34] so some patients with systemic amyloidosis will by chance have an MGUS that is coincidental to their non-AL amyloidosis.[35] Conversely, not all cases of true AL amyloidosis have a paraprotein detectable by conventional serum protein electrophoresis.

Screening for the immunoglobulin light chain abnormality that accompanies AL amyloidosis requires a combination of serum protein immunofixation electrophoresis, urine protein immunofixation electrophoresis and the serum free light chain (FLC) assay. A clonal abnormality (serum paraprotein in ∼80%, urine Bence-Jones proteinuria in ∼70% or abnormal FLC ratio in ∼75%[36]) will be apparent in 95–99% of cases, and omission of one of these testing modalities produces a significant reduction in sensitivity.[27, 36] Nevertheless, 2–5% of true AL cases have no detectable paraprotein or FLC abnormality because of inability of these assays to detect very low levels of monoclonal FLCs among the normal polyclonal background.

Bone marrow aspirate and biopsy for quantitation of plasma cell burden is recommended only for those with suspected AL amyloidosis; additionally, occasionally the trephine biopsy may be a useful site in which to search for vascular or interstitial amyloid if its presence has not yet been proven. Cytogenetics (metaphase and/or fluorescence in situ hybridisation) are recommended only if there is a significant plasma cell burden. Bone imaging (skeletal survey, MRI or positron emission tomography, as appropriate) is indicated only if a plasma cell dyscrasia is identified and, along with other markers of aggressive plasma cell behaviour like hypercalcaemia and the plasma cell burden, allows one to determine whether the AL amyloidosis is due to an MGUS or myeloma.

The inflammation underlying AA amyloidosis can have many causes (Table 4).[2] A careful clinical enquiry for evidence of chronic inflammatory arthropathy or bowel disease, infection and hereditary fever syndromes should be made. Systemic inflammation is usually present for many years before the clinical onset of AA amyloidosis, and its likelihood is, in general, related to the severity and longevity of the inflammatory process. The normal inflammatory protein serum amyloid A (SAA) is the amyloid-forming protein, and ideally, serum levels should be measured. However, in practice, this assay is not routinely available and the C-reactive protein is an adequate substitute. Serial measurements may be required if AA is suspected. In a small but well-recognised group of patients with proven AA amyloidosis, there is no identifiable inflammatory disease clinically.

Table 4. Underlying disorders associated with AA amyloidosis[2]
Chronic inflammatory arthritis60%
Rheumatoid arthritis33%
Juvenile idiopathic arthritis17%
Chronic sepsis15%
Bronchiectasis5%
Injection drug use4%
Complications of paraplegia (infected pressure sores, urinary infection)2%
Osteomyelitis1%
Tuberculosis1%
Periodic fever syndromes9%
Familial Mediterranean fever5%
Crohn disease5%
Other chronic inflammation6%
Malignancy1%
Castleman disease2%
Vasculitis1%
Unknown6%

Role of genetic screening

Although uncommon, a genetic cause for amyloidosis should be considered in all patients, as many cases have no family history because of incomplete penetrance, unrecognised onset or death from other causes in previous generations. The exclusion of hereditary amyloidoses is often very useful in helping solidify the diagnosis of AL amyloidosis. Testing for most hereditary forms of amyloidosis is available in Australia (ATTR, AFib, ApoA1, ALys. Contact: Associate Professor David Booth, Pathology West, Department of Immunology, Level 2, ICPMR Building, Westmead Hospital; Email: david.booth@sydney.edu.au) and New Zealand (ATTR, AFib. Contact: Canterbury Health Laboratories, Christchurch, NZ) or internationally (contact: Professor Philip Hawkins, National Amyloidosis Centre, London, UK; Email: p.hawkins@ucl.ac.uk). Blanket testing of all patients is inappropriate on grounds of cost and long turnaround times but should be considered where (i) hereditary amyloidosis is suspected or (ii) one cannot rule it out on phenotypic grounds. For example, standard practice should be to test for TTR mutations in isolated cardiac or mixed cardiac/nerve cases and for fibrinogen mutations in isolated renal amyloidosis. Alternatively, however, investigation of unknown cases is more appropriately done by direct identification of the target protein, for example by tandem mass spectrometry.

Immunohistochemistry and other histological techniques

In most centres, apart from renal biopsies where there is the added luxury of a fresh frozen sample for direct immunofluorescence, the subtyping of amyloid is usually performed by immunohistochemical staining (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues. Many laboratories report stains using antibodies to SAA, TTR, and kappa and lambda FLC. Although IHC, when well performed on optimally fixed and processed tissue, can be reliable in a significant percentage of cases,[18] problems are not infrequently encountered[35, 37, 38] even in the hands of international centres performing the technique frequently. Common problems include weak staining for κ and λ light chains in AL amyloidosis,[35] false-positive staining of light chains in non-AL amyloid,[37] background staining of light chains because of ‘locking-in’ of serum proteins during fixation,[39] and false-positive staining for TTR in AL amyloid.[38] False-negative and -positive results are common, and positive results for several different stains on a single biopsy specimen may be observed. Although other more reliable histological techniques are available, such as direct immunofluorescence on fresh frozen tissue samples[39] and immuno-EM[40] (only available in Italy), recent developments in the use of mass spectroscopy (see later discussion) and its availability in both Australia and New Zealand mean that it has become the investigation of choice to resolve difficult cases where IHC is inconclusive or atypical. Clinicians treating patients with amyloid should be aware of the limitations of IHC and discuss difficult cases with their nearest amyloid referral centre. Where appropriate, a joint decision to refer biopsies for mass spectroscopy is the best approach.

Occasionally the histological appearance of the amyloid deposits may give a clue to the amyloid subtype. For example, in AFib, the distribution of the amyloid is somewhat unique possibly because of the relative selectivity of this type of amyloid for glomeruli. In this condition, the glomeruli have a characteristic appearance, being markedly enlarged by amyloid, while the renal interstitium and blood vessels contain almost no amyloid at all.[6] It should also be noted that potassium permanganate pretreatment followed by Congo red staining is unreliable for subtyping and should no longer be used.[18]

Tandem mass spectrometry

In the past decade, new methods have been developed that have enabled the use of FFPE tissues to be used in the study of proteomics by mass spectrometry, the first report being in 2005.[41] Laser capture microdissection has been used to procure the usually small amounts of amyloid deposits and to ensure that the sample is relatively pure, with minimal protein contaminants present from the normal surrounding tissues.[42] Advances in high-performance liquid chromatography and mass spectrometry technologies have led to greater sensitivity for low-level samples. This is particularly salient to the subtyping of amyloid, as in some biopsy samples, the amyloid deposits may only be present in a few blood vessels. Early reports of the use of laser capture microdissection and tandem mass spectrometry in clinical biopsy specimens suggest that this method will soon become the gold standard for accurate subtyping of amyloidosis.[43] The technique can be used to identify any of the amyloid subtypes whether they be acquired or hereditary variants. Currently, tandem mass spectrometry-based techniques are available in the research setting in Australia (contact: Dr Patricia Renaut, Department of Anatomical Pathology, Princess Alexandra Hospital, Brisbane; Patricia_Renaut@health.qld.gov.au) and New Zealand (contact: Dr Hugh Goodman, Haematology Department, Waikato Hospital, Hamilton; hugh.goodman@waikatodhb.health.nz).

Key points related to subtyping of amyloid

  • Correct subtyping of amyloidosis is critical in all cases
  • The combination of a monoclonal immunoglobulin abnormality on serum or urine testing, and amyloid on a biopsy is highly suspicious but not diagnostic of AL amyloidosis
  • Potassium permanganate pretreatment followed by Congo red staining is unreliable for subtyping and should no longer be used
  • Subtyping of amyloid using immunohistochemistry of biopsies may give false-positive or discrepant results
  • Subtyping of amyloid using mass spectroscopy is becoming the new gold standard and is available in Brisbane and Auckland on request
  • Assessment of clinical phenotype, associated diseases, immunohistochemistry, genetic testing and tandem mass spectrometry will allow identification of the type of amyloid (Fig. 3).
Figure 3.

Overall approach to amyloid subtyping. κ, kappa light chain; λ, lambda light chain; AFib, amyloid derived from fibrinogen ; AL, amyloid derived from immunoglobulin light chain; ATTR, amyloid derived from transthyretin; BNP, brain natriuretic peptide; CRP, C-reactive protein; ECG, electrocardiogram; FLC, serum free light chain assay; IHC, immunohistochemistry; LFT, liver function test; MRI, magnetic resonance imaging; PCD, plasma cell dyscrasia; SAA, serum amyloid A protein; SPEP, serum protein electrophoresis with immunofixation; TandemMS, tandem mass spectrometry; TTR, transthyretin; UPEP, urine proten electrophoresis with immunofixation.

Overall approach to amyloid subtyping

An approach to subtyping is summarised in Figure 3. As tandem mass spectrometry is not yet routinely available and immunohistochemistry has limitations, amyloid subtyping currently requires a multidisciplinary synthesis of information. Fortunately, however, the multitudinous amyloid subtypes present as a smaller number of common scenarios.

First, how much proof is needed to diagnose AL amyloidosis? This is the commonest conundrum encountered as the diagnosis must be sufficiently certain to justify proceeding with chemotherapy. Most patients with amyloidosis and a paraprotein do have AL, but unfortunately, the absence of a routinely available high-quality confirmatory test means that assumptions must be made. Cases with a phenotype that is highly likely to be AL (e.g. the combination of heart, kidney and nerves, or a feature such as macroglossia or factor X deficiency) when present with a paraprotein can be treated as AL without further testing as long as other clues (e.g. family history, concurrent inflammatory illness) are not present. Cases with an ambivalent phenotype, even when a plasma cell dyscrasia is present, must be further investigated.

Second, isolated renal amyloidosis is common. Most are AL and have a plasma cell dyscrasia, but there may be clues to other subtypes that should be pursued. Fibrinogen gene testing should be considered, and many patients will need tandem mass spectrometry for a firm diagnosis.

Third, isolated cardiac amyloidosis, with or without neuropathy (peripheral and/or autonomic), can be a difficult problem, particularly in the elderly. In youngerpatients, the differential diagnosis is between AL and hereditary mutations of TTR (ATTRm) that is readily solved by TTR gene testing. In those over 60 years, both senile systemic/cardiac amyloidosis (wild-type TTR; ATTRwt) and (potentially coincidental) MGUS become increasingly common, and therefore, in the elderly with a cardiac +/− neuropathic phenotype, AL and ATTRwt cannot be distinguished reliably without tandem mass spectrometry.

There are many other patterns of phenotype, histology, associated diseases and other data, some of which may allow amyloid subtyping with confidence. Tandem mass spectrometry is an invaluable tool for diagnosis in cases of uncertainty. In the event of difficult cases, the authors are always happy to be contacted for advice.

Conclusion

Amyloidosis is a rare but important disease that must be suspected, especially when typical clinical presentations are present. Early diagnosis allows access to all treatment options before advanced organ dysfunction develops. Accurate subtyping is central to the management of amyloidosis. In certain cases, the diagnosis may require a series of advanced diagnostic techniques and experience to identify correctly the causative protein. However, accurate subtyping of the amyloid is critical for correct management of the patient and should be sought in all cases so that all patients can have their amyloid protein identified to a high level of confidence.

Ancillary