淀粉样物质检测与纤维分型近观：CAP 指南与阅片提示Close-up on amyloid detection and fibril typing

Amyloidosis has become a treatable disease—if the fibril type is identified accurately and quickly.

That and the variability seen in its workup and diagnosis were the impetus for the recently published CAP amyloidosis guideline (Miller DV, et al. Arch Pathol Lab Med. 2026;150[3]:179–193).

The co-chairs of the guideline expert panel provided, in a CAP25 session, what they described as their own tips and tricks for amyloid detection and fibril typing, reported here.

There are at least 42 amyloid subtypes, said panel co-chair Billie S. Fyfe, MD, professor in the Department of Pathology and Laboratory Medicine at Rutgers Health Robert Wood Johnson Medical School. “And with mass spectrometry, more amyloid types are being identified each year.”

The fibril type dictates the treatment, said co-chair Dylan V. Miller, MD, professor of pathology at the University of Utah School of Medicine and chair of the CAP Immunohistochemistry Committee. “In my mind, that sounds a lot like a predictive marker test,” he said, “like PD-L1 and HER2 and others.”

If left untreated, 25 percent of patients with immunoglobulin light chain (AL) amyloidosis die within six months of diagnosis, and a similar percentage with amyloid transthyretin (ATTR) amyloidosis die within 24 months of diagnosis. All forms of amyloidosis are underdiagnosed (Gertz MA, et al. JAMA. 2020;324[1]:79–89).

Guideline recommendations (“guideposts”)

Recommendation 1. In patients with suspected systemic amyloidosis, pathologists may screen cytology specimens (conventional smears and/or cell blocks) of aspirated abdominal fat for detection of amyloid. Individual laboratories should determine and optimize the best preparation methods and ancillary testing techniques should be validated on cytologic material. If cytologic smears only are prepared in the absence of a cell block, the ability for further testing, including subtyping, is limited.

There are data to support screening of cytology specimens by conventional smears or cell blocks, Dr. Miller said. “If you’re not using a tissue-based method, you should be sure the method you’re using works for detecting amyloid.” If using an air-dried smear, “you should know when you do Congo red on that smear you are able to detect amyloid in your lab.” With a limited sample like an air-dried smear, fibril typing will not be able to be performed. “There may be a need to get more tissue for doing that important part of the diagnostic workup.”

Recommendation 2. When evaluating specimens for the presence of amyloid, pathologists should use the Congo red staining method. Other methods can be used but should be validated against Congo red or electron microscopy and must show equivalency.

Recommendation 3. When assessing Congo red histochemistry, pathologists may add fluorescence microscopy with the tetramethylrhodamine isothiocyanate/Texas red filter to increase sensitivity for amyloid detection. “You don’t need to buy a fluorescence scope to do this,” Dr. Miller said, “but if you have one, this is a helpful thing you can do to improve sensitivity of your Congo red interpretation.”

Recommendation 4. In patients with amyloidosis for whom therapy is being considered, mass spectrometry should be used to identify the fibril protein type in order to optimize diagnostic yield and the use of tissue. In renal amyloidosis, amyloid fibril typing may often be accomplished successfully by immunofluorescence, though reflex to MS-based proteomics should be performed in difficult or equivocal cases.

Good practice statements

• When specimens are received for detection of systemic amyloidosis, pathologists should evaluate for the presence of amyloid using validated method(s), and the validated method(s) used should be identified in the pathology report. For a fat pad or other surrogate biopsy that is negative, “be very clear that that doesn’t exclude the possibility of amyloid in a target organ or elsewhere in the body.”
• If a clinical concern for amyloidosis persists after a negative biopsy from a surrogate site but the potentially affected organ (or organs) was not sampled, then suggesting a biopsy of the potentially affected organ(s) and/or recommending suitable archived specimen(s) to evaluate is appropriate.
• In patients with amyloidosis who are being considered for therapy, pathologists should determine the fibril protein type.

IHC can be effective for fibril identification, but panels are only as good as optimization of each assay; it is a “closed-ended exercise” versus mass spectrometry. Immunoelectron microscopy combines confirming definitive amyloid fibrils with gold-labeled monoclonal antibodies. Mass spectrometry on paraffin tissue is the gold standard, with very high sensitivity and specificity, detecting the amyloid type regardless of fibril peptide—and even multiple overlapping types.

Practical tips for slide reading

Check the on-slide tissue control (Fig. 1). Ideally it should show good staining of what is supposed to be amyloid and have a negative component.

“Not everything that’s red on a Congo red is positive for amyloid,” Dr. Miller said (Fig. 2). “This is not immunohistochemistry, it’s not a trichrome. And Congo red tends to be very prone to overstaining and nonspecific staining, so do not just look at a slide at 2× and say it is Congo red positive even though it’s pretty red.”

He always begins with the H&E. For him to call something amyloid, it has to look like amyloid on the H&E—though that is not sufficient. Prototypical renal glomeruli show pink, glasslike, smooth or waxy material without cells embedded (Fig. 3). “So if the H&E looks like it could be amyloid, and the Congo red is hitting everywhere where it could be amyloid, then I’m pretty suspicious.”

By contrast, in diabetes (Fig. 4), cellularity and fibrosis are visible where there is pink material; fibrotic areas show ropy collagen or fine fibrillar texture rather than smooth glass. “You shouldn’t see any fibrillar material or texture on the H&E in true amyloid.” Heavy overstaining that does not match H&E candidates is unreliable. Amyloid loves vessel walls, but nonspecific staining of smooth muscle/elastic must be excluded.

Edge and stripe artifacts and heavy regional overstaining are common (Fig. 5). “Overstaining and artifacts are almost the rule now, at least in my lab,” Dr. Miller said. They use the Dako (Agilent) Artisan autostainer for Congo red and are “never going back to hand-staining.”

Nonspecific congophilia is a pitfall: chondroid, erythrocytes, skeletal and smooth muscle, and elastic tissue in vessels; dense collagen (blue-white birefringence) and fibrin can also pick up Congo red nonspecifically. “Be careful if you’re seeing Congo red in vessels, and it looks a little smooth and amorphous, that it’s not just elastic tissue.”

Fluorescence can highlight Congo red positivity (Fig. 6). The guideline prefers “characteristic birefringence” rather than only “apple green”; orangish hues can be seen and should match nonpolarized and polarized Congo red findings.

Heart and fat pad biopsies (Dr. Fyfe)

Cardiologists are now diagnosing ATTR on the basis of scans; patients may present with MGUS and an equivocal scan. Heart biopsies are characteristically fairly straightforward but “are sometimes now a bit trickier.”

Fig. 7 shows H&E (“definitely amyloid, maybe subtle”) and trichrome of an endomyocardial biopsy from a patient with MGUS. Hearts with amyloidosis can also have fibrosis; trichrome for amyloid deposits is supposed to look a little bluish-gray (Fig. 8).

Another MGUS case shows subtle Congo red on H&E (Fig. 9) but strong vascular staining on TRITC fluorescence (Fig. 10). Her institution freezes tissue for light-chain immunofluorescence; she also sends mass spectrometry. Fig. 11: lambda MGUS with lambda IF positive and also kappa staining; MS confirmed AL (lambda) amyloidosis.

“Fat pads are another thorn in people’s side,” Dr. Fyfe said. In Fig. 12 Congo red is focally positive, with birefringence and bright orange fluorescence—“So it was diagnostic to me”; the patient went on to AL amyloidosis.

Nontargeted tissues and localized amyloid

The guideline does not suggest staining every carpal tunnel or lumbar stenosis specimen with Congo red, but awareness matters when clinicians raise concern.

The guideline addresses only systemic amyloidosis; localized amyloidosis is seen in larynx, skin, bowel (injury), and rarely urinary tract. A potentially systemic fibril type in a biopsy does not always mean systemic disease (e.g., pulmonary amyloidoma AL lambda). Repeated abdominal fat injections (e.g., long-term insulin) can produce localized amyloid-like deposits. GLP-1 agonist–type injections: “possible but probably needs more time to evolve.”

Therapies (context)

AL therapies include plasma cell– and immune-directed agents (daratumumab, cyclophosphamide, dexamethasone, belantamab mafodotin; proteasome inhibitors; ASCT; newer anti-amyloid antibodies). ATTR therapies differ (RNA silencing of hepatic TTR production; tafamidis stabilizer; seeding inhibitors and antibodies under study).

Amy Carpenter is CAP TODAY senior editor.