How can the different types of amyloid be differentiated?

How to Differentiate Between Different Types of Amyloid

The differentiation of amyloid types requires a two-step approach: first, confirm amyloid presence with Congo red staining showing apple-green birefringence under polarized light, then definitively type the amyloid using immunohistochemistry or mass spectrometry on tissue biopsy. 1

Initial Detection and Confirmation

Congo red staining remains the gold standard for detecting amyloid deposits, demonstrating characteristic apple-green birefringence under polarized light microscopy. This has approximately 100% specificity and sensitivity for detecting amyloid deposits 2. Once amyloid is confirmed, the critical next step is determining which type you're dealing with.

Algorithmic Approach to Typing Amyloid

Step 1: Screen for Monoclonal Protein

• Obtain serum immunofixation, urine immunofixation, and serum free light chains (FLC) 3
• Check the FLC ratio (normal range 0.26-1.65 by Binding Site assay, or 0.31-1.56 by Siemens) 1
• If monoclonal protein is present: Suspect AL amyloidosis and proceed to tissue typing
• If no monoclonal protein is detected: ATTR amyloidosis becomes more likely

Step 2: Cardiac-Specific Pathway (If Cardiac Involvement Present)

For patients WITHOUT monoclonal protein:

• Radionuclide scintigraphy (PYP, DPD, or HMDP scan) alone can diagnose ATTR cardiac amyloidosis without requiring endomyocardial biopsy 2
• This is a critical point: the absence of monoclonal protein plus positive cardiac scintigraphy is diagnostic for ATTR-CM

For patients WITH monoclonal protein (even MGUS):

• Endomyocardial biopsy is necessary to definitively distinguish between AL and ATTR cardiac amyloidosis 2
• This is because both conditions can coexist, and treatment differs dramatically

Step 3: Tissue Biopsy and Typing

Biopsy site selection:

• Target organ biopsy (e.g., endomyocardial biopsy if heart involved) provides highest sensitivity and specificity 2
• Surrogate biopsies can be attempted first: abdominal fat pad (45% sensitive for ATTRm, only 15% for ATTRwt) or bone marrow 2, 3
• Critical caveat: A negative fat pad or bone marrow biopsy does NOT rule out amyloidosis—proceed to target organ biopsy if clinical suspicion remains high 2

Amyloid typing methods (perform on positive biopsy):

• Immunohistochemistry: Stain for AA, ATTR, A-lambda, A-kappa, and A-beta-2-microglobulin 1
• Mass spectrometry: Increasingly preferred as it provides definitive protein identification 1
• Both methods are required regardless of biopsy site to confirm the amyloidosis subtype

Step 4: Additional Testing Based on Type

If ATTR amyloidosis is confirmed:

• Perform TTR gene sequencing in ALL patients, even without family history, to distinguish ATTRv (hereditary/variant) from ATTRwt (wild-type) 2, 4
• Over 120 TTR mutations exist with variable penetrance 1
• If variant detected, genetic counseling for family members is indicated 2

If AL amyloidosis is confirmed:

• NT-proBNP (>99% sensitivity when ≥332 ng/L) and troponins for staging 1
• Bone marrow biopsy to assess plasma cell burden

Historical Methods (Less Commonly Used Now)

Potassium permanganate (KMnO4) pretreatment:

• AA amyloid loses Congo red affinity after KMnO4 treatment ("sensitive") 5, 6
• AL and ATTR amyloid retain Congo red affinity ("resistant") 5, 6
• This method has been largely superseded by immunohistochemistry and mass spectrometry but can be useful when modern techniques are unavailable

Common Pitfalls to Avoid

1. Do not rely on fat pad biopsy alone for ATTR amyloidosis—its low sensitivity (especially for ATTRwt at 15%) means negative results are unreliable 2

2. Do not skip TTR gene sequencing even in elderly patients without family history—ATTRwt can occur sporadically and treatment differs from ATTRv 2, 4

3. Do not diagnose ATTR-CM by scintigraphy if ANY monoclonal protein is present—endomyocardial biopsy is mandatory in this scenario 2

4. Do not assume a single type of amyloidosis—patients can have multiple types simultaneously (e.g., AL and ATTR, or localized plus systemic forms) 7

5. Do not delay treatment waiting for perfect typing—early intervention dramatically improves outcomes, with 80% 5-year survival in early-stage disease versus <30% in advanced disease 8