Familial Renal Amyloidosis Workup

• No blood or urine test result is diagnostic of amyloidosis, but lab findings that exclude chronic inflammation or a monoclonal gammopathy in a patient with renal amyloid accumulation support the possibility of FRA. Lab tests also have a vital role in evaluating and monitoring amyloidotic organ function.
• Once the creatinine clearance has fallen to less than 20%, progression to end-stage renal failure is almost inevitable, although the rate of decline often does not accord with predictions and may be remarkably slow. On the other hand, step-wise deteriorations in renal function occur quite frequently, even in the absence of any identifiable intercurrent renal insult such as dehydration, infection, or venous thrombosis.
• Liver function test results tend to remain normal until the liver has been extensively infiltrated by amyloid, and even marked hepatomegaly may be accompanied by only a modest elevation in serum alkaline phosphatase. Liver function in those with FRA is often well preserved for decades, and elevations of serum bilirubin and transaminase levels occur at a very late stage. A bilirubin value of just twice the upper limit of normal is associated with a very poor prognosis and incipient liver failure.
• Hematological indices and coagulation tend to be unremarkable, although a hyposplenic picture can occur. Occult GI blood loss should be considered in patients with anemia that is not secondary to renal impairment.

• Anatomical imaging modalities (eg, plain radiography, computed tomography [CT] scan, magnetic resonance imaging [MRI], ultrasonography)

  • Typically, these yield nonspecific findings in patients with systemic amyloidosis. However, a study by Barreiros et al suggests that ultrasonography can reveal signs of amyloidosis in various organs.^[5] In an examination of 30 patients with systemic amyloidosis, including 19 suffering from familial amyloid polyneuropathy, the investigators found the following ultrasonographic indications of amyloidosis:
    • Heart - Myocardial thickness, pericardial and pleural effusion, and typical echorich subendocardial depositions
    • Liver and spleen - Spontaneous subcapsular hemorrhages
    • Intestine - Inhomogeneous, patchy-like depositions
    • Kidney - Somewhat unspecific results in this organ
  • Amyloidotic organs may be enlarged in the late stage of the disease, but kidney size varies and may be normal or even small at presentation.
  • Amyloid deposits are rich in calcium, and areas of calcification may develop.
  • Radionuclide tracers used for bone scintigraphy occasionally localize in amyloidotic organs.
• SAP component scintigraphy

  • This was introduced in 1987 and is a sensitive, specific, and noninvasive method of quantitatively imaging amyloid deposits in vivo.
  • All amyloid fibrils bind the normal plasma protein SAP by virtue of a specific calcium-dependent ligand-protein interaction.
  • In patients with amyloidosis, iodine I¹²³ –labeled SAP localizes rapidly and specifically to the amyloid deposits.
  • The technique has a high diagnostic sensitivity and is the only method available for serial monitoring of the progression or regression of amyloid throughout the body.
  • SAP scintigraphy is eminently suitable as a screening test in patients thought to be at risk for systemic amyloid deposition, including those with known amyloidogenic mutations. However, the technique is not yet available commercially.
  • Serial SAP scans have shown that accumulation of amyloid in patients with FRA tends to be much slower compared to patients with acquired AA and AL types, and progression may not be evident, even over the course of a decade.
  • In all types of acquired and hereditary amyloidosis that have been studied, SAP scans have also shown that amyloid deposits are often cleared gradually when the supply of amyloid fibril precursor proteins can be reduced.
  • Scintigraphic image findings are depicted below. Progression of amyloid deposits in a patient with amyloidosis associated with fibrinogen A alpha-chain Glu526Val. These serial posterior, whole-body, scintigraphic images were obtained following intravenous injection of iodine-123 (123I)–labeled human serum amyloid P component into a 48-year-old man with hereditary amyloidosis associated with fibrinogen A alpha-chain Glu526Val in whom asymptomatic proteinuria had been identified. Both parents were alive and well and older than age 80 years. The scan at diagnosis (left) showed modest abnormal uptake into renal amyloid deposits, which increased at follow-up 3 years later (right). The remainder of the image represents a normal distribution of tracer throughout the blood pool. Regression of amyloidosis associated with fibrinogen A alpha-chain Glu526Val following hepatorenal transplantation. The pictures are serial anterior, whole-body, scintigraphic images obtained following intravenous injection of iodine-123 (123I)–labeled human serum amyloid P component into a patient with amyloidosis associated with fibrinogen A alpha-chain Glu526Val. Prior to hepatorenal transplantation (left), heavy amyloid deposition was present in an enlarged liver and spleen. No amyloid deposits were identified in a follow-up study obtained 42 months after hepatorenal transplantation (right); only a normal distribution of tracer is present throughout the blood pool. Regression of amyloidosis associated with apolipoprotein AI Gly26Arg following hepatorenal transplantation. These serial anterior, whole-body, scintigraphic images were obtained following intravenous injection of iodine-123 (123I)–labeled human serum amyloid P component into a patient with hereditary amyloidosis associated with apolipoprotein AI Gly26Arg. Prior to hepatorenal transplantation (left), heavy amyloid deposition was present in the liver, obscuring the kidneys. Two years after combined hepatorenal transplantation (right), a follow-up scan was normal, showing tracer distributed evenly throughout the background blood pool, including the transplanted organs. Splenic amyloid deposits that were evident initially in posterior scans had regressed at follow-up.
• Echocardiography

  • Significant cardiac amyloid deposition is relatively unusual in patients with FRA, especially in patients with lysozyme and fibrinogen types, but confers a poor prognosis when it is present.
  • Amyloid causes diastolic dysfunction with well-preserved contractility until a very late stage.
  • Cardiac amyloidosis is best evaluated by a combination of echocardiography, ECG, and measurement of NT-pro BNP.
  • The classic findings with 2-dimensional Doppler echocardiography are concentric biventricular wall thickening, increased myocardial echodensity, thickened but pliable valves, and a restrictive filling pattern.
  • ECG findings may be normal in patients with substantial cardiac amyloidosis, but reduced voltages, pathological Q waves (ie, pseudoinfarct pattern) in the anterior chest leads, and conduction abnormalities usually signify advanced disease.

• DNA analysis

  • DNA analysis is mandatory in all patients with systemic amyloidosis who cannot be confirmed absolutely to have the AA or AL type. Appreciating that the presence of a chronic inflammatory disease or a monoclonal gammopathy may be incidental is important.
  • Numerous mutations have been identified in most of the genes associated with hereditary amyloidosis, and new variants are being found regularly. Therefore, performing gene sequencing is better than using methods such as restriction fragment length polymorphism analysis, which is directed at particular known mutations.
  • The results of DNA analysis are not, by themselves, definitive proof of the presence of amyloid or the amyloid fibril type, and these findings must be interpreted in light of other clinical and histologic findings.
• Fibril protein sequencing

  • In cases in which identifying the amyloid fibril type by more conventional means is not possible, isolation of amyloid fibrils from a sample of fresh amyloidotic tissue enables amino acid sequencing of the fibril subunit peptide.
  • This requires technical expertise and is time consuming but can be achieved using very small tissue samples. It is the most definitive method for typing amyloid deposits.

• The definitive diagnosis of amyloid accumulation requires histologic confirmation; however, biopsy procedures carry an increased risk of hemorrhage in patients with amyloidosis, and bleeding may be substantial and even life-threatening in 5% of patients who undergo biopsies. This is due to the increased fragility of amyloidotic blood vessels and the reduced elasticity of severely affected organs.
• Less-invasive alternatives include fine-needle aspiration of subcutaneous fat and rectal or labial salivary gland biopsy. In experienced hands, these screening biopsies can yield positive results in as many as 80% of cases; however, in routine practice, sensitivity is only approximately 50%. Also, fat aspirates are usually not suitable for immunohistochemical typing.

Many cotton dyes, fluorescent stains such as thioflavine-T, and metachromatic stains have been used, but Congo red staining and its resultant green birefringence when viewed with high-intensity cross-polarized light has the best specificity and is the criterion standard histochemical test for amyloidosis. The stain is unstable and must be freshly prepared at least every 2 months. A section thickness of 5-10 µm and inclusion in every staining run of a positive-control tissue containing modest amounts of amyloid are critical to ensure specificity and quality control. Other problems in histologically based diagnoses include obtaining adequate tissue samples and an unavoidable element of sampling error. Biopsies cannot reveal the extent or distribution of amyloid accumulation, and failure to demonstrate amyloid in one or even several biopsies does not exclude the diagnosis.

Although many amyloid fibril proteins can be identified immunohistochemically, the demonstration of potentially 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 protein are commercially available and always stain AA deposits. However, in patients with AL amyloid, the deposits are stainable with standard antisera to kappa or lambda only in approximately half of all cases. This is probably because the light-chain fragment in the fibrils is usually the N-terminal variable domain, which is largely unique for each monoclonal protein.

Immunohistochemistry produces variable results in patients with FRA; the staining is typically weak in patients with fibrinogen A alpha-chain amyloid but is more reliable in patients with lysozyme and apolipoprotein AI types. Including positive tissue and absorption controls in each run is vital for optimal interpretation of the results.

The appearance of amyloid fibrils in tissues under the electron microscope is not always completely specific, and, sometimes, they cannot be identified convincingly. Although electron microscopy should be more sensitive than light microscopy, it is not sufficient by itself to confirm the diagnosis of amyloidosis.

A recent advance in diagnostic techniques is the use of laser microdissection and mass spectrometry to directly identify the components of the amyloid deposits. A large, single center study has demonstrated that proteomics can be successfully used to type amyloid deposits with more accuracy than conventional immunohistochemistry.^[6]