What laboratory tests are recommended for a comprehensive amyloid workup?

Laboratory Tests for Amyloid Workup

For a comprehensive amyloid workup, obtain serum free light chains (sFLC), serum immunofixation electrophoresis (SIFE), and urine immunofixation electrophoresis (UIFE) simultaneously—never rely on serum/urine protein electrophoresis (SPEP/UPEP) alone, as these miss up to 50% of cases due to the characteristically low monoclonal protein levels in AL amyloidosis. 1

Essential Laboratory Tests

Monoclonal Protein Screening (Required for All Suspected Cases)

• Serum free light chain (sFLC) assay: Measures kappa and lambda free light chains independently and calculates the kappa:lambda ratio, providing superior sensitivity compared to standard electrophoresis 1
• Serum immunofixation electrophoresis (SIFE): More sensitive than SPEP for identifying and typing monoclonal immunoglobulins 1, 2
• Urine immunofixation electrophoresis (UIFE): Essential because some patients have negative serum studies but positive urine findings 1, 2

Critical pitfall: SPEP/UPEP should never be used to exclude AL amyloidosis due to significantly lower sensitivity compared to immunofixation, particularly given the low monoclonal protein burden typical of AL amyloidosis (unlike multiple myeloma) 1

Performance Characteristics of Monoclonal Protein Testing

• Free light chain assay specificity: 90% for predicting AL amyloid type with positive predictive value of 87% 3
• SPEP/UPEP/IFE specificity: Only 75% with positive predictive value of 74%, making it a poor predictor when used alone 3
• Combined serum and urine IFE: Achieves 81.3% sensitivity and 97.6% specificity for renal AL amyloidosis in patients over 60 years 4

Important caveat: The specific IFE system and reagents used can significantly affect detection rates—up to 50% of samples testing negative on one IFE system (Epalyzer2) tested positive when retested on another system (HYDRASYS 2), particularly in patients with low involved free light chain (iFLC) or low difference between involved and uninvolved free light chain (dFLC) values 5

Additional Required Tests

Bone Marrow Evaluation

• Bone marrow biopsy: Demonstrates clonal proliferation of lambda or kappa-producing plasma cells (or B cells), required to confirm AL amyloidosis diagnosis 1
• Plasma cell quantification: Evaluates percentage of plasma cells and identifies atypical lymphoid or lymphoplasmacytic aggregates 2
• Myeloma FISH panel: Characterizes plasma cell dyscrasias 2

Key point: Bone marrow biopsy has 69% sensitivity for finding amyloid deposits in systemic AL amyloidosis, but mass spectrometry typing is mandatory because over 10% of patients with monoclonal gammopathy can have ATTR deposits in bone marrow 1

Exclusion of Multiple Myeloma and Lymphoproliferative Disorders

• Bone marrow examination excludes multiple myeloma or B-cell lymphoproliferative disorders (rarely B-cell lymphoma) 1
• Approximately 10-15% of multiple myeloma patients also have AL amyloidosis, making this distinction clinically critical 6

Tissue Diagnosis and Typing

Biopsy Approach

Two acceptable strategies exist 1:

1. Direct biopsy of affected organ (definitive approach)
2. Surrogate site biopsy first (bone marrow and/or abdominal fat aspiration), then proceed to affected organ biopsy if Congo red staining is negative

Abdominal Fat Pad Biopsy Sensitivity

• AL amyloidosis: 76-95% sensitivity (84% for AL cardiac amyloidosis) 1, 7
• ATTRv (hereditary) cardiac amyloidosis: 45% sensitivity 1, 7
• ATTRwt (wild-type) cardiac amyloidosis: Only 15% sensitivity 1, 7

Critical decision point: Fat pad biopsy is inadequate for ATTR cardiac amyloidosis screening—if ATTR is suspected and monoclonal protein screen is negative, proceed directly to bone scintigraphy or endomyocardial biopsy 7

Amyloid Typing (Gold Standard)

• Mass spectrometry (LC-MS/MS): Gold standard with 88% sensitivity and 96% specificity for identifying the amyloidogenic protein 1, 7, 6
• When LC-MS/MS unavailable: Transfer Congo red-positive samples to experienced reference laboratory for definitive typing 1
• Alternative methods: Immunohistochemistry or immunogold immunoelectron microscopy (less reliable than mass spectrometry) 1, 7

Mandatory in specific scenarios: Tissue biopsy precursor typing is particularly critical when monoclonal gammopathy of undetermined significance (MGUS) coexists with suspected ATTR cardiac amyloidosis, as Congo red staining alone cannot distinguish between types 1

Special Considerations for Cardiac Amyloidosis

When Monoclonal Protein is Detected

• Endomyocardial biopsy required: Definitively distinguishes AL from ATTR cardiac amyloidosis when any monoclonal protein is present (even MGUS), as both can coexist and bone scintigraphy alone is insufficient 1, 7
• Cardiac biopsy preferred: In suspected concomitant systemic AL and ATTR cardiac amyloidosis (MGUS with abnormal nuclear scintigraphy) 1

Organ-Specific Biomarkers for Monitoring

• Cardiac involvement: NT-proBNP, troponin, electrocardiography, echocardiography 1
• Renal involvement: Creatinine, 24-hour urine protein electrophoresis 1
• Hepatic involvement: Liver enzymes, alkaline phosphatase 1

Algorithmic Approach Summary

1. Initial screening: Order sFLC, SIFE, and UIFE simultaneously (never SPEP/UPEP alone) 1
2. If monoclonal protein detected: Proceed with bone marrow biopsy and tissue biopsy (fat pad or affected organ) 1
3. If monoclonal protein negative: Consider ATTR amyloidosis—proceed with genetic testing for TTR mutations and/or bone scintigraphy 7
4. All Congo red-positive biopsies: Mandate mass spectrometry typing (LC-MS/MS) to identify precursor protein 1
5. Confirm AL amyloidosis: Requires both tissue amyloid deposits AND demonstration of plasma cell disorder 1

Final critical point: Hematology collaboration is essential whenever monoclonal protein testing is abnormal to distinguish spurious findings from kidney dysfunction, true MGUS, AL amyloidosis, or multiple myeloma 1