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Ways to Confirm Amyloidosis Diagnosis - An Overview

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Amyloidosis is a rare disease caused by the deposition of amyloid protein and can be confirmed in various ways. Read below to learn more.

Written by

Dr. Neha Rani

Medically reviewed by

Dr. Sugandh Garg

Published At June 11, 2024
Reviewed AtJune 11, 2024

What Is Amyloidosis?

Amyloidosis is an uncommon illness characterized by the accumulation of aberrant (abnormal) amyloid protein deposits within the body. These deposits may impact the kidneys, spleen, brain, heart, and other organs. Amyloidosis can affect one or more organs in a person.

Amyloidosis may develop on its own or as a complication of another illness. Sometimes, the source is unknown, while other times, it can be due to a gene mutation.

What Are the Various Ways to Confirm the Diagnosis of Amyloidosis?

Amyloidosis occurs when aberrant protein deposits, or amyloid fibrils, accumulate in organs or tissues, impairing their functionality and possibly resulting in death. The normal proteins turn insoluble, resulting in amyloid fibrils. Although the symptoms of various types of amyloidosis are identical, it is becoming increasingly evident that the kind of precursor protein that determines the amyloid fibrils can predict the particular disease.

Amyloidosis is usually diagnosed by detecting amyloid deposits in a tissue sample through biopsy. The only kind of amyloidosis that can occasionally be detected without a tissue sample is cardiac transthyretin amyloidosis. Scintigraphic imaging can diagnose patients with symptoms and suggestive findings from echocardiography or cardiac MRI (magnetic resonance imaging), provided no plasma cell disease exists.

Determining the protein responsible for amyloidosis is crucial, as distinct forms necessitate distinct therapies. Patients may be prescribed the incorrect chemotherapy if the incorrect kind is detected.

The various ways to confirm the diagnosis of amyloidosis are:

  • Tissue biopsy.

  • Immunohistochemistry.

  • Immunofluorescence.

  • Immunoelectron microscopy.

  • Mass spectrometry-based proteomic typing.

How Is Amyloid Deposition Identified in Tissue Samples?

  • On sections stained with hematoxylin and eosin (HE), amyloid deposits appear shapeless and pinkish. However, this technique is not specific to amyloid; similar appearances can also be caused by sclerosis (abnormal hardening of body parts or tissue) or hyaline alterations.

  • Thioflavin dyes, mainly Congo red, confirm the presence of amyloid. Amyloid stained with Congo red gives off a salmon pink or red appearance under a standard microscope. The confirmation sign is the presence of specific birefringence under cross-polarized light when the amyloid deposits appear apple-green. However, they may also exhibit other colors, such as yellow-green or blue-green, to apple-green, partly due to additional birefringence from other structures in the light path, such as glass slides.

  • While effective for detecting amyloid deposits, Congo red staining may also stain other structures, such as the elastic laminae of arteries, eosinophils, and myelomatous casts. However, under cross-polarized light, these structures will appear white. It is crucial to follow staining protocols, as over-staining can yield false positive results, while using aged stains may lead to false negatives.

  • Using thin sections raises the risk of missing amyloid deposits, so thick sections of at least 5 µm (micrometer) are recommended. Additionally, novel fluorescent dyes have promising potential for identifying and typing amyloid deposits.

What Are the Various Biopsy Sites for Amyloidosis?

  • Biopsy of a Clinically Involved Organ: The most accurate way to diagnose amyloidosis is to biopsy a clinically affected organ, such as the kidney or heart. This procedure can identify any concomitant diseases and provide additional tissue for amyloid typing later on.

  • Biopsy of a Surrogate Site: Organ biopsies carry higher risks, even if they can be safely performed in well-chosen amyloidosis patients. It takes technical expertise to perform organ biopsies, and patients may experience increased discomfort. So, the first diagnostic strategy frequently advised, particularly in cases where AL amyloidosis is suspected, is the biopsy of a surrogate location, which offers a reasonable level of diagnostic sensitivity. However, a negative biopsy from a surrogate site does not necessarily rule out amyloidosis. Hence, a biopsy of an afflicted organ should be performed if clinical suspicion is maintained.

  • Abdominal Subcutaneous Fat Aspiration (ASFA): ASFA is commonly used to diagnose amyloidosis. It is easy to perform at the bedside and produces no discomfort. However, it might not always give enough tissue, which could result in negative results or insufficient material for amyloid typing. It is advised to aspirate from several locations on the abdomen wall and to use a big needle to increase precision.

  • Rectal Biopsy: Systemic amyloidosis is diagnosed by rectal biopsy with the abdominal subcutaneous fat sample. Its effectiveness has been disputed. Since amyloid usually builds up in the rectum's muscularis mucosae and submucosa, a biopsy that obtains mucosal tissue may miss the diagnosis. Rectal biopsies can be painful, necessitate patient preparation, and carry certain hazards, including bleeding and perforation. Recent research shows rectal biopsy yields are generally low, particularly in the event of negative ASFA results. As a result, the rectal biopsy is no longer frequently advised as the main technique for diagnosing amyloidosis.

  • Bone Marrow Biopsy: Patients suspected of having systemic AL amyloidosis should get a bone marrow biopsy evaluation and a core biopsy. It is recommended that bone marrow biopsy be combined with ASFA to boost detection rates up to 90 percent. However, the type of amyloid found in the bone marrow is not always the same as in AL amyloidosis. Other forms of amyloidosis that impact the heart or are caused by inflammation may also be connected.

  • Biopsy of Other Surrogate Sites: A small portion of the salivary gland is extracted from the inside of the mouth, close to the lower canine teeth, during a minor salivary gland biopsy (MSLB), which is effective in diagnosing systemic amyloidosis. After the surgery, some people could have transient problems, including tingling or edema (swelling). Skin and gingival biopsies have also been used to make diagnoses, but they are less sensitive and may cause greater patient discomfort. Skin biopsies only provide positive results in roughly 50 percent of cases of systemic amyloidosis.

What Are the Various Methods Used to Determine the Amyloid Type?

  • Immunohistochemistry: Immunohistochemistry (IHC) uses antibodies to target particular protein epitopes for amyloid typing. It is readily available, reasonably priced, and frequently detects the amyloidogenic protein. To reduce false positives and negatives, its dependability depends on using an approved antibody panel. The major drawbacks are that background staining may be caused by normal proteins containing targeted epitopes or by non-immunological binding when using immunohistochemistry for amyloid type. Since antibodies may bind to normal immunoglobulins, this makes diagnosing AL amyloid more difficult. False positive results may result from trapped serum proteins contaminating amyloid plaques. Furthermore, amyloidogenic proteins may lose epitopes during fibril formation, decreasing their antibody reactivity.

  • Immunofluorescence: Similar to immunohistochemistry, immunofluorescence (IF) uses fluorescently marked antibodies against target epitopes that can be seen under a fluorescence microscope. IF frequently uses frozen sections to avoid the autofluorescence present in paraffin sections. Fixation-related problems such as plasma protein entrapment and antigenic site change are circumvented by this method. As a result, IF is less susceptible than IHC to background staining and restricted antibody reactivity. IF has been used to characterize amyloid fibril proteins, but the results have been inconsistent, which is similar to the variability shown with IHC.

  • Immunoelectron Microscopy: Immunoelectron microscopy (IEM) combines electron microscopy with IHC to detect proteins within amyloid fibrils as determined by ultrastructural analysis. By utilizing the high-resolution electron microscopy capabilities and the accuracy of antibody-based tagging with gold particles, this approach improves the specificity of protein characterization within amyloid plaques.

  • Mass Spectrometry-Based Proteomic Typing: Due to the limitations of antibody-based methods, direct chemical characterization of proteins within amyloid deposits is crucial. Laser capture microdissection (LCM) followed by tandem mass spectrometry (MS) is a powerful approach. LCM allows precise isolation of amyloid deposits, minimizing contamination. Subsequent MS analysis enables the identification of amyloid fibril proteins with high sensitivity and specificity, even those of low abundance or novel ones. This method overcomes the shortcomings of antibody-based techniques, offering valuable insights into amyloid composition and structure.

Conclusion

Histological identification of amyloid on tissue biopsy is necessary to diagnose amyloidosis. Although biopsies of clinically affected organs produce the best diagnostic yield, biopsies of surrogate sites, like bone marrow, small salivary glands, or abdomen subcutaneous fat, can also safely and comfortably confirm the presence of amyloid. Though more recent techniques are not yet available, the recommended method for verifying amyloid accumulation remains Congo red staining. Further subtyping is needed to determine the particular amyloidogenic protein, which can be done with the help of immunohistochemistry, immunofluorescence, immunoelectron microscopy, and mass spectrometry-based proteomic typing.

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Dr. Sugandh Garg
Dr. Sugandh Garg

Internal Medicine

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