Immunoglobulin-Related Amyloidosis Workup

Once the diagnosis of L chain–type amyloidosis has been established (see Biopsy), the clinician should perform laboratory studies to observe for abnormalities that are commonly found in these patients (eg, impairment in kidney function or coagulation) and to evaluate for possible multiple myeloma.

Tests for monoclonal immunoglobulin

Monoclonal immunoglobulin L chain, the cardinal laboratory finding in L chain–type amyloidosis, is detected by electrophoresis or immunoelectrophoresis in the serum or the urine of 80-90% of patients. This percentage reflects the limit to the sensitivity of routine laboratory testing rather than the biology of L chain–type amyloidosis. Because plasma cells in bone marrow (or occasionally in other sites) synthesize immunoglobulin L chains, which are deposited in various organs, the L chains must travel through the bloodstream. Thus, in theory, if a sufficiently sensitive assay were used, monoclonal serum L chains or L-chain fragments would be detected in all patients.

Overall survival is related to the free monoclonal light-chain concentration in the serum, independent of other risk factors, with higher concentrations associated with shorter survival. ^[11]

The concentration of normal immunoglobulin is often decreased, such as in multiple myeloma. The combination of hypogammaglobulinemia and proteinuria should suggest a diagnosis of L chain–type amyloidosis or monoclonal immunoglobulin deposition disease (MIDD). In contrast, renal amyloid of the amyloid A type is usually associated with hypergammaglobulinemia related to persistent inflammation and interleukin-6 (IL-6) production.

A study by Dispenzieri et al showed that the absolute value of immunoglobulin free light chain (FLC) as a precursor protein of amyloid is prognostic in patients with primary systemic amyloidosis undergoing peripheral blood stem cell transplantation (PBSCT). ^[12] Risk of death was significantly higher in patients with higher baseline free light chains, and normalization of the free light chain level after PBSCT predicted for both organ response and complete hematologic response.

Blood counts

The complete blood cell count (CBC) is usually unremarkable. Functional asplenism may occur, leading to mild thrombocytosis and Howell-Jolly bodies in the peripheral blood.

Absolute lymphocyte count recovery at day 15 (ALC-15) after autologous stem cell transplantation seems to be a powerful prognostic indicator for overall survival and progression-free survival. ^[13] An ALC-15 of 500 or greater is associated with significantly improved clinical outcomes.

Coagulation studies

Many clotting system abnormalities have been described in L chain–type amyloidosis. Occasionally, coagulopathy and prolongation of the prothrombin time (PT) and activated partial thromboplastin time (aPTT) arise because of the binding of a clotting factor (most often factor X) to the amyloid deposits. Acquired factor X deficiency is difficult to correct because infused factor X is cleared quickly from the circulation.

Elevation in tissue and urine plasminogen activators and a decrease in tissue plasminogen activator inhibitor, leading to hyperfibrinolytic states, have also been reported.

Urinary protein

When L chain–type amyloidosis involves the kidneys, proteinuria is invariably present. One third to one half of patients excrete at least 1 gram of protein per day in the urine, predominantly albumin.

The 24-hour urinary protein level can be monitored serially to evaluate the response to chemotherapy. A decrease in protein excretion may be associated with improvement in response to treatment.

Liver and kidney function studies

Liver function abnormalities are rare, even in cases with massive deposition. Rarely, extensive liver involvement can lead to decreased levels of vitamin K-dependent clotting factors.

On kidney function studies, severe azotemia is a late manifestation of renal L chain–type amyloidosis and is less common than proteinuria. However, mild elevation of the serum creatinine level (at least 2 mg/dL) is often present.

Bone marrow examination

Approximately 40% of patients have more than 10% plasma cells in the bone marrow. L-chain immunophenotyping of the marrow, even in the absence of increased numbers of plasma cells, usually exhibits the distortion in the k:l ratio, reflecting the L-chain type of the amyloid precursor.

Paiva et al reported that the use of multiparameter flow cytometry immunophenotyping to demonstrate plasma cell clonality in patients with amyloid-positive tissue staining could help in the differentiation of light chain amyloidosis from other forms of amyloidosis, and that the quantitative distribution of monoclonal and normal plasma cell populations may be useful in establishing the prognosis of individual patients with respect to overall survival. ^[14] However, the report did not specifically evaluate responses to therapy in general or any specific treatment. This kind of prognostic information requires a much larger patient cohort.

Cardiac imaging

Cardiac deposition is the most serious complication of L chain–type amyloidosis. Cardiac involvement should be assessed and monitored by means of imaging studies. No noninvasive test is sufficiently sensitive or specific to definitively diagnose cardiac amyloidosis, although two-dimensional echocardiography (2-D echo) and electrocardiography (ECG), particularly when combined, can strongly suggest cardiac amyloidosis. Echocardiography in combination with cardiovascular magnetic resonance (CMR) imaging can also be used to diagnose and prognosticate cardiac amyloidosis. ^[15]

Other cardiac imaging studies, such as computed tomography (CT) scanning and nuclear scintigraphy, are of less value than ECG and echocardiography.

Echocardiography

The most useful noninvasive diagnostic test for cardiac amyloidosis is echocardiography, which enables the 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 it is highly suggestive when present.

L chain–type amyloid deposits in the heart occur in the ventricular interstitium, leading to thickening of the ventricular walls and intraventricular septum without an increase in intracardiac volume. Evaluation of diastolic function with 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 the disease course.

Other echocardiographic findings include valvular thickening and insufficiency and atrial enlargement. Atrial thrombosis has also been described. Combining ECG and echocardiography appears to provide the most diagnostic value.

Cardiac magnetic resonance imaging

CMR has high spatial resolution and provides tissue characterization. Late gadolinium enhancement (LGE) patterns on CMR are highly sensitive and specific for cardiac amyloidosis and may even precede morphologic increases in left ventricular wall thickness. ^[16] However, the area of involvement can also be patchy, diffuse, or transmural. Transmural enhancement has been associated with a poorer prognosis than other patterns of enhancement. ^[17]

Bone imaging

As in any patient with a plasma cell dyscrasia, patients with L chain–type amyloidosis should have a skeletal survey that includes the skull, the entire spine, and the pelvis. Any bony pain that develops can result from plasma cell infiltration; therefore, obtain radiographs of any area where pain develops.

Chest radiography

In systemic L chain–type amyloidosis, amyloid may be deposited in any part of the respiratory tree, from the nasopharynx to the pulmonary alveoli. Involvement is often asymptomatic, although alveolar or diffuse interstitial involvement can cause dyspnea. Chest radiographs reveal a reticular nodular pattern or interstitial infiltration.

Nuclear medicine

Scintigraphy with radioiodine-labeled serum amyloid pentagonal component (SAP) is used in Europe for evaluating the total body burden of amyloid and is a sensitive, noninvasive means of diagnosing amyloid deposits in most organs. Serial studies are useful for monitoring response to therapy. The technique is not useful for diagnosing or monitoring cardiac amyloid, because the concentration of the radiolabeled agent in the intracardiac blood pool obscures the weaker signal from the labeled molecule bound to myocardial amyloid. However, SAP scintigraphy is not approved by the US Food and Drug Administration because the SAP is isolated from human sources. ^[18]

Amyloid imaging agents that do not involve human materials include a synthetic heparin-binding peptide, p5, radiolabeled with technetium 99m (^99m Tc-p5). A study by Wall et al that compared radioiodine-labeled SAP with ^99mTc-p5 concluded that ^99mTc-p5 is an effective amyloid-imaging radiotracer in the murine model of amyloidosis, and may be rapidly translated for imaging patients with visceral amyloidosis. ^[19]

In an earlier phase I study, Wall and colleagues used a radiolabeled monoclonal amyloid antibody to visualize amyloid deposits in patients with AL amyloidosis. Nine of the 18 subjects showed striking uptake of reagent in liver, lymph nodes, bone marrow, intestine, and spleen (but not kidney or heart). These authors concluded that this method could be used to identify candidates for passive immunotherapy using the chimeric form of the antibody, which has been shown to produce marked regression of human light-chain–associated tumors in mice. ^[20]

Obtaining a biopsy sample 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 kappa and lambda L chains proves the diagnosis of L chain–type amyloidosis (as opposed to other types of amyloidosis, which have a similar appearance after hematoxylin and eosin or Congo red staining) (see Amyloidosis).

In monoclonal immunoglobulin deposition disease (MIDD), the immunoglobulin deposits do not bind Congo red stain, they do not contain P component or other components of amyloid fibrils, and (unlike in amyloidosis) they are not fibrillar. MIDD occurs most frequently in the kidneys and the heart. Nodular glomerulosclerosis observed on routine histologic examination in the absence of diabetes mellitus suggests MIDD. The pathologic diagnosis of nonamyloid MIDD depends on the identification of immunoglobulin deposits in tissues via immunostaining. MIDD may be underdiagnosed because immunostaining is not routinely performed.

The clinical pathologic feature and diagnostic criteria of tongue amyloidosis is important. In one study of 25 patients pathologically diagnosed as having tongue amyloidosis, although none had an enlarged tongue, hematoxylin and eosin and immunohistochemical staining were employed to detect the amyloid deposition on the tongue, with amyloid depositions in the basement membrane, muscle cell, vessel wall, and nerve fiber. ^[21]

Immunohistochemical study demonstrated kappa light-chain deposition in 64% of cases, and lambda light-chain deposition in 36%. ^[21] Thus, biopsy is an important means for the diagnosis of early tongue L chain–type amyloidosis, and the wide variety of amyloid light chains is helpful in the differential diagnosis (see DDx).

The classic ECG finding in patients with cardiac amyloidosis 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. A variety of arrhythmias are observed and can be life threatening.

Amyloidosis of all types is definitively diagnosed by a positive Congo red stain of a biopsy specimen. For many years, a biopsy of the rectum was the procedure of choice. However, it is known that capillaries in subcutaneous fat are frequently involved. These capillaries can provide sufficient tissue for the diagnosis of amyloidosis, and further analysis with immunostaining and, in some cases, amino acid sequence analysis.

Currently, aspiration of subcutaneous abdominal fat is a simple and fast method for obtaining a specimen to detect systemic amyloidosis, with a routine sensitivity of 80%. If analysis of aspirated fat tissue yields negative results, the additional value of a subsequent biopsy of the rectum is negligible.

Thus, obtaining a biopsy from the organ with the most severe clinical involvement is not always necessary. However, a biopsy from an organ with impaired function, such as a kidney or the heart, definitively establishes a cause-and-effect relationship between the organ dysfunction and the amyloid deposition.

The gold standard of cardiac amyloidosis diagnosis is myocardial biopsy. However, this procedure is invasive and may fail to detect cardiac amyloidosis if the biopsy sample is taken from a region of the myocardium unaffected by amyloidosis. The type of cardiac amyloidosis may be distinguished by taking a sample of the biopsy tissue and analyzing it by mass spectrometry. In clinical practice, most patients have tissue diagnosis from fat pad or kidney biopsies. ^[17]

L chain–type amyloid deposition in the peripheral nerves leads to axonal degeneration of the small nerve fibers, which leads to polyneuropathy. The diagnosis can often be made through findings from a biopsy of the sural nerve, although the deposits may be proximal to the sural nerve and, therefore, not found in the biopsy sample.

Obtaining a kidney biopsy sample is rarely necessary, but findings in such cases include deposits in the glomerular mesangium and, later, along the basement membrane.

Other potential biopsy sites include the salivary glands, the stomach, and the bone marrow.

Avoid obtaining a percutaneous liver biopsy. Such biopsies are contraindicated in the presence of coagulopathy. Severe and even fatal bleeding has occurred in this setting.

Immunostaining

After Congo red staining is used to establish a diagnosis of amyloidosis, the specific type of amyloidosis is determined by immunostaining a biopsy specimen using commercially available, specific antisera against k and l chains.

Do not assume that the amyloid is of the L-chain type based on indirect tests, such as serum or urine protein electrophoresis or immunofixation, because monoclonal proteins are common in the elderly population and may be present as incidental findings in patients with other types of amyloidosis.

Distinguishing between L chain–type amyloidosis and TTR cardiac amyloidosis on clinical grounds alone is particularly difficult. Without immunologic identification of the deposited protein, an incorrect presumptive diagnosis of L chain–type amyloidosis could lead to ineffective and perhaps harmful treatment.