Transthyretin-Related Amyloidosis Workup

Updated: Jan 26, 2017
  • Author: Jefferson R Roberts, MD; Chief Editor: Emmanuel C Besa, MD  more...
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Workup

Approach Considerations

Diagnostic tests for transthyretin (TTR) amyloidosis are listed in the table below (adapted from Ando et al [45] ).

Table. Diagnostic tests for transythyretin (TTR) amyloidosis (Open Table in a new window)

Method Material Sensitivity Specificity Purpose
Pathologic        
Congo Red Tissue Medium/High High Detecting amyloid deposits
BSB, FSB dyes Tissue High  Medium Detecting amyloid deposits
Electron microscopy Tissue Medium High Confirming amyloid fibrils
Immunohistochemistry with anti-TTR antibodies Tissue High Medium/High Detecting TTR deposits
Genetic        
PCR-RFLP DNA High High Detecting predicted mutations in the TTR gene
Real-time PCR (melting curve analysis) DNA High High Detecting predicted mutations in the TTR gene
PCR-SSCP DNA Medium Medium Screening for unknown mutations in the TTR gene
Sequencing DNA High High Detecting unknown mutations in the TTR gene
Mass Spectrometry (MS)        
MALDI-TOF MS, ESI-MS Serum protein Medium/High Medium Detecting variant TTR
FT-ICR MS Serum protein Medium/High Medium/High Detecting variant TTR
SELDI-TOF MS Serum protein Medium/High Medium Detecting variant TTR
LC-MS/MS Tissue Medium Medium Identifying precursor proteins of amyloid fibrils, including variant TTR

BSB = 1-Bromo-2,5-bis(3-carboxy-4-hydroxystyryl)benzene; ESI = electrospray ionization; FSB = 1-Fluoro-2,5-bis(3-carboxy-4hydroxystyryl)benzene; FT-ICR = Fourier transform ion cyclotron resonance; LC-MS/MS = liquid chromatography–tandem mass spectrometry; MALDI-TOF = matrix-assisted laser desorption/ionization time-of-flight; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism; SELDI-TOF = surface enhanced laser desorption/ionization–TOF; SSCP = single-strand conformation polymorphism 

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Laboratory Studies

Nonspecific findings found in different types of amyloidosis include the following:

  • Normochromic normocytic anemia
  • Electrolyte abnormalities secondary to heart failure or malabsorption
  • Varying degrees of proteinuria, albuminuria, azotemias, and diminished glomerular filtration rate in patients with renal deposition [45]
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Biopsy

Amyloidosis (of all types) is diagnosed definitively based on demonstration of Congo red binding material in a biopsy specimen. (See the image below.)

Congo Red staining of a cardiac biopsy specimen co Congo Red staining of a cardiac biopsy specimen containing amyloid, viewed under polarized light.

For many years, rectal biopsy was the favored procedure when systemic amyloidosis was suspected. Currently, the capillaries in subcutaneous fat are known to be involved often in TTR-related amyloidosis (ATTR) and in some other types of systemic amyloidosis; therefore, subcutaneous fat aspiration often provides sufficient tissue for diagnosing amyloid, as well as for further studies such as immunostaining. On the other hand, biopsy of an organ with impaired function, such as the heart or gastrointestinal tract, has the advantage of definitively establishing a cause-and-effect relationship between organ dysfunction and amyloid deposition.

The sensitivity of detecting ATTR varies by site, as follows [16] :

  1. Labial salivary gland biopsy: 91%
  2. Sural nerve biopsy: 80%
  3. Abdominal fat pad biopsy: 14-83%

ATTR deposition in the peripheral nerves leads to axonal degeneration of the small nerve fibers, causing polyneuropathy. Diagnosis can often be made with sural nerve biopsy, although the deposits may be proximal to the sural nerve and therefore not found in biopsy samples.

Other potential biopsy sites include the following:

  • Myocardium
  • Stomach
  • Rectum
  • Other organ suspected of heavy involvement

Amyloid should not be assumed to be of the TTR type based solely on the Congo red staining and clinical picture. After Congo red staining establishes a diagnosis of amyloidosis, the specific type of amyloidosis must be determined with immunostaining of a biopsy specimen using commercially available antiserum against TTR. Control antisera against other types of amyloid precursors, including immunoglobulin light chains and amyloid A protein, should also be performed to confirm staining specificity. Even patients known to carry a TTR variant should ideally have the diagnosis confirmed with immunostaining to rule out the possibility of a different type of amyloidosis.

Distinguishing between ATTR and AL cardiac amyloidosis on clinical grounds alone is particularly difficult. Without immunologic identification of the deposited protein, an incorrect diagnosis of ATTR in a patient with AL, or the reverse, could lead to ineffective or harmful treatment. Mass spectroscopy can also be used to determine the protein subunit and classify the disease as immunoglobulin light-chain amyloidosis or ATTR. [20]

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Imaging Studies

N/A

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Cardiac Imaging

Cardiac deposition is, in many patients, the most serious complication of ATTR. For that reason, cardiac involvement usually should be assessed and monitored by imaging studies. Echocardiograms, cardiac magnetic resonance imaging (MRI), and scintigraphy with bone tracers can all help to diagnose infiltrative cardiomyopathy. [16, 49]

Echocardiography enables visualization of increased ventricular wall thickness, increased septal thickness, and an appearance of granular "sparkling." This finding is neither sensitive nor specific enough to be diagnostic but is highly suggestive when present.

Amyloid deposits in the heart occur in the ventricular interstitium, leading to thickening of the ventricular walls and interventricular septum without an increase in the intracardiac volume. Evaluation of diastolic function by Doppler echocardiography reveals impaired ventricular relaxation early in the course of disease, which progresses to short deceleration. The ejection fraction is preserved until late in disease. [20]

Other echocardiographic findings include the following:

  • Valvular thickening
  • Valvular insufficiency
  • Atrial enlargement
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Other Tests

Other cardiac testing

The classic finding on electrocardiography is a low-voltage QRS complex in the limb leads, resulting from replacement of normal cardiac tissue by nonconducting amyloid material. In some cases, loss of anterior forces suggests anteroseptal infarction that is not confirmed at autopsy. Various arrhythmias are observed and can be life threatening. [15]

The prevalence of low QRS voltages at the time of diagnosis has been found to be lower than in light chain amyloidosis (AL), despite the finding, in some studies, of greater myocardial infiltration in TTR-related forms. [21]

Holter monitoring and intracardiac electrophysiology study are helpful to detect conduction disorders. [16]

Nerve conduction studies

In patients with amyloid neuropathy, serial nerve conduction studies can be useful for objectively monitoring the course of disease and for assessing response to treatment such as liver transplantation. [22]

Genetic studies

In patients with progressive, length-dependent axonal neuropathy predominantly involving small nerve fibers, genetic testing for TTR gene mutations should be performed during the initial diagnostic workup, to prevent serious consequences from delayed diagnosis. [23] Genetic studies to look for a TTR variant can be helpful in many patients with ATTR, particularly in younger patients not known to belong to a kindred carrying a defined TTR variant. These studies generally are not available through routine clinical laboratories.

One approach is to perform polymerase chain reaction (PCR) testing to look for known, common TTR variants. This approach is most useful if the likely TTR variant can be surmised on the basis of the clinical history and genetic background of the patient. These studies are performed by PCR amplification of regions of the TTR gene followed by digestion with restriction enzymes.

If a TTR variant is suspected but initial screening results for a few common known variants are negative, more comprehensive analysis for a TTR variant can be performed. Either the protein can be isolated from the serum and studied using methods such as electrospray ionization mass spectrometry (ESIMS) or the gene can be studied by PCR and such methods as single-strand conformation polymorphism analysis and/or direct sequencing.

Determination of whether a TTR variant is present is important because the treatment options for variant-sequence ATTR differ from those for normal-sequence ATTR. Information about a TTR variant also can be of use to other family members at risk. 

Ophthalmological assessment

Ophthalmological assessment is warranted to identify any ocular manifestations of TTR-FAP, which may include the following:

  • Keratoconjunctivitis sicca
  • Secondary glaucoma
  • Vitreous opacities
  • Pupillary abnormalities

Radiolabeled P-component scanning

Radiolabeled P-component scanning is available in a few European centers. Where it is available, radiolabeled P-component scanning is a very useful means of evaluating the total body burden of amyloid and is a sensitive noninvasive means of diagnosing amyloid in most organs. Serial studies are useful for monitoring the response to therapy in many settings.

One drawback of P-component scanning is that it is not useful for diagnosing or monitoring cardiac amyloid, because the concentration of label in the intracardiac blood pool obscures the weaker signal from the labeled molecule bound to myocardial amyloid.

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Histologic Findings

Biopsy of an affected organ followed by routine hematoxylin and eosin staining reveals homogeneous interstitial eosinophilic material. Amyloid material stained with Congo red and viewed under polarized light appears bright green. Specific staining with antibodies against TTR proves the diagnosis of ATTR, as opposed to other types of amyloidosis that have similar appearance after hematoxylin and eosin or Congo red staining.

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Procedures

N/A

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Staging

Once the diagnosis has been made, the neuropathy stage and systemic extension of the disease should be determined to direct the course of management. The three stages of ATTR-FAP severity are graded according to the patient’s walking disability and degree of assistance required, as follows [16, 17] :

Stage 0: Asymptomatic carrier of a known ATTR mutation

Stage I: Sensory polyneuropathy; preserved walking capacity without the need for a walking stick

Stage II: Progressive walking disability; ambulatory, but requires assistance, one to two walking sticks or crutches required

Stage III: Wheelchair bound or bedridden

 

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