Intestinal amyloidosis: diagnosis and confirmation

Intestinal amyloidosis refers to the deposition of amyloid fibrils in the gastrointestinal tract wall, often as part of systemic amyloidosis. Clinical manifestations are often nonspecific, so the diagnosis is almost never made based on symptoms or blood tests alone without morphological confirmation. [1]

The main principle of confirmation is the same for all variants: first, it is necessary to prove the presence of amyloid in the tissue, and then determine the protein it is composed of. This is critical, because treatment strategies differ radically for immunoglobulin light chain amyloidosis, amyloidosis associated with chronic inflammation, and hereditary variants. [2]

For the intestines, "double" accuracy is especially important, as the disease can mimic inflammatory bowel disease, celiac disease, chronic infections, ischemic colitis, functional disorders, and drug-induced enteropathies. Errors in typing can lead to inappropriate treatment and wasted time. [3]

Modern diagnostics are based on a sequence of clinical suspicion, targeted biopsy, Congo red staining with polarization, then amyloid typing by immunohistochemistry or mass spectrometry, followed by an assessment of systemic involvement and a search for the underlying cause of a specific type of amyloidosis. [4]

Table 1. What is considered confirmation of diagnosis in practice [5]

Level	What should be obtained	What does this prove?
1	Amyloid in biopsy, Congo red plus characteristic fluorescence in polarized light	The fact of amyloidosis
2	Amyloid protein typing (e.g., light chains, serum amyloid A, transthyretin, and others)	What form of amyloidosis and what basic therapy is required?
3	Confirmation of systemic and organ damage plus search for the protein source	Complete diagnosis and treatment plan

When should intestinal amyloidosis be suspected?

Suspicion is heightened when chronic intestinal symptoms are combined with signs of a systemic process. Gastrointestinal amyloidosis is typically characterized by persistent diarrhea, malabsorption, weight loss, nausea, pain, bloating, and dysmotility, including pseudo-obstruction. [6]

The key clinical sign is intestinal symptoms against the background of a known predisposing situation. For type AL amyloidosis, there are often markers of plasma cell disease and damage to the heart, kidneys, and peripheral nerves. For type AA amyloidosis, there is usually a long-term chronic inflammatory disease and signs of chronic inflammation. [7]

Significant manifestations of protein-losing enteropathy include edema, low albumin levels in the absence of pronounced nephrotic syndrome and severe cirrhosis, and chronic diarrhea. Amyloid can be deposited perivascularly and in the submucosa, disrupting the barrier and lymphatic drainage, which contributes to protein loss. [8]

Additional clues include bleeding and anemia, especially with multiple submucosal hemorrhages and vascular fragility. The endoscopic picture can be variable, so if suspicion persists, a biopsy with proper staining becomes crucial. [9]

Table 2. Symptoms and clinical clues that increase the likelihood of intestinal amyloidosis [10]

Manifestation	What's alarming	Why is this important?
Diarrhea for more than 4 weeks, poor response to standard therapy	persistence and progression	amyloidosis often mimics other causes of chronic diarrhea
Weight loss, signs of malabsorption	deficiencies, steatorrhea, weakness	small intestinal lesion and malabsorption
Edema plus low albumin	protein-losing enteropathy	a typical scenario in case of intestinal wall damage
Bleeding, iron deficiency anemia	repeated episodes or occult bleeding	vascular fragility and mucosal infiltration
Signs of systemic amyloidosis	kidneys, heart, nerves, liver	intestinal symptoms are often part of a systemic process

Pre-biopsy screening: what really helps guide the diagnosis

Initial testing does not confirm amyloidosis on its own, but it helps assess its severity, identify complications, and select the appropriate diagnostic approach. A complete blood count, biochemistry panel with albumin and electrolytes, iron levels, inflammatory markers, and assessment of kidney and liver function are helpful. [11]

In cases of diarrhea and suspected malabsorption, assessment of protein and fat losses is appropriate, as are baseline stool tests to rule out infections, depending on the clinical context. These steps are necessary to avoid missing more common causes of chronic diarrhea and interpreting every finding as amyloidosis. [12]

If the clinical picture resembles systemic AL amyloidosis, it is advisable to immediately search for a monoclonal protein in parallel with a gastrointestinal examination: serum and urinary immunofixation, as well as serum free light chain analysis. These tests do not replace an intestinal biopsy, but they significantly increase the pre-test probability and speed up accurate typing. [13]

If systemic amyloidosis is suspected, a biopsy of accessible tissue, such as a subcutaneous fat aspiration, is often useful for initial confirmation. This is less invasive than an organ biopsy, but sensitivity depends on the type of amyloid and the total amyloid burden, so a negative result does not rule out the disease and, if highly suspected, requires a biopsy of the affected organ. [14]

Table 3. Primary diagnostic package for suspected intestinal amyloidosis [15]

Target	Examination	What does it give?
Assess complications	complete blood count, albumin, electrolytes, liver and kidney function	anemia, hypoalbuminemia, electrolyte disturbances
Support the suspicion of a systemic process	inflammatory markers, assessment of associated organs	helps to distinguish between the inflammatory background and severity
Don't miss the AL option	serum and urine immunofixation, free light chains	search for a monoclonal protein source
Rapid confirmation of systemic amyloidosis less invasively	subcutaneous fat aspiration with Congo red staining	screening for systemic amyloidosis, but not always negative
Decide on a bowel biopsy	gastroenterologist consultation plus endoscopy plan	selection of biopsy area and volume

Confirmation in the intestine: endoscopy and biopsy

The gold standard for confirming gastrointestinal amyloidosis is mucosal biopsy with Congo red staining and polarized light evaluation. Radiologic studies may suggest lesions or dysmotility complications, but do not alone confirm amyloidosis. [16]

The choice of endoscopic approach depends on symptoms. If upper intestinal lesions are suspected, esophagogastroduodenoscopy with biopsies is performed; if colonic symptoms are present, colonoscopy with biopsies is performed. In systemic amyloidosis, deposits can be multifocal, so multiple biopsies are preferred even with minimal visual changes. [17]

The diagnostic yield of biopsies is often considered high in the duodenum, followed by the stomach and colon, although results vary by population and technique. Studies of gastrointestinal biopsies have shown a high frequency of deposit detection in the duodenum and rectum, so these sites are often considered a priority in cases of unexplained diarrhea and suspected systemic processes. [18]

A separate practical issue concerns the depth of deposits. For some forms of amyloidosis, deposits may predominate in the submucosa and around vessels, so a superficial "thin" biopsy can sometimes be falsely negative. This is one argument in favor of multiple, high-quality samples, and, if suspicion persists, repeat endoscopy with refined technique. [19]

If suspicion is very high and biopsies are negative, three factors should be reconsidered: the accuracy of Congo red staining and reading, the adequacy of the specimen, and the feasibility of biopsy of other tissue, including subcutaneous fat or an affected extraintestinal organ. Reviews have noted that some patients may present with a clinical picture of gastrointestinal amyloidosis despite negative biopsies, necessitating a more comprehensive diagnostic approach rather than automatically ruling out the disease. [20]

Table 4. Where to take biopsies if intestinal amyloidosis is suspected [21]

Situation	Priority biopsy area	Why
Chronic diarrhea, malabsorption	duodenum plus stomach	often high diagnostic yield in the upper sections
Blood in stool, anemia, changes in stool	large intestine plus rectum	vascular deposits and bleeding are possible
Suspected systemic process without clear localization	multiple biopsies from several segments	amyloidosis can be multifocal
Negative results with high suspicion	repeat biopsies to clarify depth and quality	False negatives are possible due to submucosal distribution

Morphological confirmation and typing of amyloid

The classic morphological criterion is Congo red staining with a characteristic "apple-green" fluorescence under polarized light. This remains the reference criterion, but the quality of the result depends on the preparation of the specimen and the experience of the interpreter; therefore, in borderline cases, re-examination in a specialized laboratory is warranted. [22]

Congo red staining answers the question "is there amyloid?" but provides little information on "what kind of amyloid." Once deposits are identified, fibrillar protein typing is required, as the same pattern in the intestine can be seen with AL, AA, ATTR, and other rare types, and treatment depends on the type. [23]

In practice, typing can be performed using immunohistochemistry with a panel of antibodies; however, this method requires experience and sometimes yields ambiguous results due to background staining and cross-reactions. Therefore, proteomic typing with laser microdissection and mass spectrometry, which allows for the recognition of a wide range of amyloid proteins in a single analysis, is increasingly being considered as the preferred approach. [24]

Mass spectrometric typing is well suited to both tissue biopsies and subcutaneous fat aspirates, and studies have demonstrated high sensitivity and specificity for the diagnosis and classification of systemic amyloidosis. This method is particularly valuable when immunohistochemistry is inconclusive or clinical features are inconsistent with the suspected type. [25]

If hereditary amyloidosis is suspected, typing is supplemented by genetic testing, as specific mutations and family counseling are important for a number of hereditary variants. Denying a hereditary variant based on clinical features alone is risky if typing reveals a protein associated with hereditary forms. [26]

Table 5. Methods of amyloid confirmation and typing: advantages and limitations [27]

Method	What confirms	Strengths	Restrictions
Congo red plus polarization	presence of amyloid	accessibility, classic standard	technical errors and weak illumination are possible
Immunohistochemistry with a panel of antibodies	type of amyloid	more accessible than proteomics	ambiguous colors, requires examination
Immune electron microscopy	type of amyloid	high specificity with good quality	limited availability
Laser microdissection plus mass spectrometry	the exact type of amyloid	one analysis for many types, high accuracy	Availability and cost depend on the laboratory
Genetic testing	hereditary variants	confirms the mutation and type of disease	does not replace tissue confirmation of amyloid

Confirmation of the systemic variant and search for the underlying cause after amyloid is found

After morphological confirmation and typing of amyloid, the next step is to prove it is a clinically significant disease, assess organ damage, and identify the source of the protein. For systemic forms, it is important not only to find amyloid in "indirect" tissue, but to link it to organ manifestations and the specific type. [28]

In AL amyloidosis, confirmation of the plasma cell clone is required. Diagnosis involves immunofixation of serum and urine, free light chain analysis, and then, if indicated, bone marrow examination and assessment of organ damage. Concurrent determination of the monoclonal protein is particularly important to avoid confusion between AL amyloidosis and the transthyretin variant or AA amyloidosis. [29]

In AA amyloidosis, the key issue is the source of chronic inflammation and long-term exposure to high levels of serum amyloid A. Therefore, after AA typing, an active inflammatory disease, chronic infection, or other condition that maintains the inflammatory response is sought, and the extent of organ damage is assessed in parallel. [30]

If typing indicates transthyretin amyloid, differentiation between the familial and wild-type variants is required, which typically involves genetic testing and assessment of cardiac and peripheral nerve involvement. To confirm the transthyretin type, tissue typing remains the primary method, while genetics refines the pattern. [31]

An assessment of systemic involvement is tailored to the clinical profile and most often includes examination of the heart, kidneys, liver, and nervous system. Specific expert imaging and biomarker algorithms exist for the heart, which help confirm involvement and indirectly support the type of amyloidosis, but amyloid typing remains key to treatment selection. [32]

Table 6. What to test after confirmation of intestinal amyloid to determine the type and underlying cause [33]

Suspect type	The main source of protein	Basic confirmation steps	What is especially important not to miss
AL	monoclonal light chains	immunofixation of serum and urine, free light chains, plasma cell clone assessment	mistaking transthyretin amyloidosis for AL and vice versa
AA	chronic inflammation with high serum amyloid A	search for active inflammatory disease, organ assessment	hidden source of inflammation and underestimation of organ damage
ATTR ancestral or wild type	transthyretin	tissue typing plus genetics if a hereditary variant is suspected	family cases and the need for genetic counseling
Other rare types	different proteins	mass spectrometric typing, sometimes genetics	wrong therapy for the wrong type

Practical confirmation algorithm

Table 7. Step-by-step diagram of “how to confirm intestinal amyloidosis” [34]

Step	Action	The result that is considered sufficient
1	Clinical suspicion based on symptoms plus assessment of systemic signs	a diagnostic hypothesis and biopsy plan were formed
2	Primary laboratory package plus parallel search for monoclonal protein in case of suspected AL	the severity was assessed and the trajectory was chosen
3	Endoscopy with multiple biopsies based on symptoms	adequate material was obtained
4	Congo red plus polarization	amyloid in tissue confirmed
5	Amyloid typing by immunohistochemistry or mass spectrometry	the protein type has been determined
6	Assessing organ damage and searching for the underlying cause by type	a complete clinical diagnosis has been formed

Common causes of false positives

A false negative is often associated with insufficient material and the peculiarities of deposit distribution across the layers of the intestinal wall, as well as with technical aspects of staining and interpretation of Congo red. If suspicion persists, a review of the slides and repeat biopsy from another area is warranted. [35]

False typing is more common with a limited immunohistochemical panel or with a nonspecific staining background. In such situations, proteomic typing using mass spectrometry reduces the risk of error and helps detect rare types of amyloid. [36]

A separate error is stopping testing after detecting a monoclonal protein in the blood without proof that it is the protein that forms amyloid. Monoclonal gammopathy can be an incidental finding, so the amyloid type must be confirmed in the tissue. [37]

Another common mistake is interpreting the discovery of amyloid in an indirect biopsy, without assessing organ involvement or seeking a cause, as a "complete diagnosis." Once amyloid is confirmed, an assessment of the clinical significance, systemicity, and cause is required; otherwise, time for therapy is lost. [38]