IMMUNOHISTOCHEMICAL PITFALLS IN AMYLOID SUBTYPING: INSIGHTS FROM A RETROSPECTIVE BRAZILIAN COHORT STUDY

Abstract

Background: Systemic amyloidosis is a potentially fatal disease caused by tissue amyloid deposition and organ dysfunction. Diagnosis is often delayed by nearly a year in low and middle-income countries. Although mass spectrometry (MS) is the gold standard for amyloid subtyping, its use is limited due to high costs and technical complexity. Immunohistochemistry (IHC) represents an alternative diagnostic method, but its diagnostic accuracy can vary significantly, particularly outside specialized laboratories with the use of commercial antibodies (AB). Objectives: To subtype amyloid by IHC and to compare it with MS, the gold standard method. Methods: In this retrospective, observational study, patients diagnosed with systemic amyloidosis from 2009 to 2018 at the HCFMUSP, a Brazilian public university hospital, were identified by database search and proactive review of biopsy records in the pathology archives. Stored biopsies were selected and included by availability to perform both IHC and MS. Amyloid tissue samples were analyzed by MS coupled with liquid chromatography following laser microdissection, thermochemical processing, and trypsin digestion. IHC panel comprised 4 commercial AB: anti-Kappa, -Lambda, -Transthyretin, -Serum amyloid A. IHC results were classified as positive (positive reaction against 1 antibody), negative (no positive reaction) or inconclusive (positive reaction against ≥2 AB of the panel). Results: From 138 patients identified with biopsy-proven systemic amyloidosis, 43 biopsies from 13 organs were included to perform both MS and IHC: 28% heart, 23% kidney, 9%, gastrointestinal tract, 7% subcutaneous fat, 7% lymph node, 5% bone marrow, 5% nerve, 5% muscle and 2% each liver, pleura, gingiva, salivary gland and tongue. Excluded biopsies were as follows: 38 insufficient for both MS and IHC, 17 external samples, 13 unavailable in the laboratory archives, 10 insufficient for MS, 9 with no remaining amyloid deposit in the stored block, and 8 insufficient for laser microdissection. The amyloid subtypes detected by MS were AL 68%, ATTR 19%, AA 10%, AFib 3%. MS results are still pending for 12 cases. IHC correctly subtyped amyloid in 10% of AL, 67% of ATTR and 67% of AA cases. Among AL patients, IHC was negative in 52%, inconclusive in 29% and positive for ATTR and AA in one case each. Among ATTR and AA patients, IHC was inconclusive in 33% each. In the AFib case, IHC ruled out AL, ATTR and AA due to negative reactions for the 4 AB in the panel. Discussion: The pitfalls of IHC in subtyping amyloid are well described in the literature. In AL, conformational changes in light chain epitopes during amyloidogenesis make IHC prone to false-negative results and background cross-reactivity to false-positives. In our cohort, IHC was not able to diagnose AL in 90% of cases, and 2 patients were misdiagnosed as ATTR and AA. In ATTR and AA, IHC failed to diagnose the subtype in one third of cases, and AFib was not diagnosed due to absence of specific AB in the panel. If adopted in clinical practice, this IHC panel would have shown poor diagnostic performance. Conclusion: Our study highlights the practical challenges of using IHC with commercial AB to subtype amyloid, reinforcing the need to expand access to MS for accurate amyloidosis diagnosis, enabling patients to receive appropriate treatment.