Ligand Profiling as a Diagnostic Tool to Differentiate Patient-Derived α-Synuclein Polymorphs.

Amyloid fibrils are characteristic of many neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. While different diseases may have fibrils formed of the same protein, the supramolecular morphology of these fibrils is disease-specific. Here, a method is reported to distinguish eight morphologically distinct amyloid fibrils based on differences in ligand binding properties. Eight fibrillar polymorphs of α-synuclein (αSyn) were investigated: five generated de novo using recombinant αSyn and three generated using protein misfolding cyclic amplification (PMCA) of recombinant αSyn seeded with brain homogenates from deceased patients diagnosed with Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). Fluorescence binding assays were carried out for each fibril using a toolkit of six different ligands. The fibril samples were separated into five categories based on a binary classification of whether they bound specific ligands or not. Quantitative binding measurements then allowed every fibrillar polymorph to be uniquely identified, and the PMCA fibrils derived from PD, MSA, and DLB patients could be unambiguously distinguished. This approach constitutes a novel and operationally simple method to differentiate amyloid fibril morphologies and to identify disease states using PMCA fibrils obtained by seeding with patient samples.

Amyloid detection in neurodegenerative diseases using MOFs.

Neurodegenerative diseases (amyloid diseases such as Alzheimer's and Parkinson's), stemming from protein misfolding and aggregation, encompass a spectrum of disorders with severe systemic implications. Timely detection is pivotal in managing these diseases owing to their significant impact on organ function and high mortality rates. The diverse array of amyloid disorders, spanning localized and systemic manifestations, underscores the complexity of these conditions and highlights the need for advanced detection methods. Traditional approaches have focused on identifying biomarkers using imaging techniques (PET and MRI) or invasive procedures. However, recent efforts have focused on the use of metal-organic frameworks (MOFs), a versatile class of materials known for their unique properties, in revolutionizing amyloid disease detection. The high porosity, customizable structures, and biocompatibility of MOFs enable their integration with biomolecules, laying the groundwork for highly sensitive and specific biosensors. These sensors have been employed using electrochemical and photophysical techniques that target amyloid species under neurodegenerative conditions. The adaptability of MOFs allows for the precise detection and quantification of amyloid proteins, offering potential advancements in early diagnosis and disease management. This review article delves into how MOFs contribute to detecting amyloid diseases by categorizing their uses based on different sensing methods, such as electrochemical (EC), electrochemiluminescence (ECL), fluorescence, Förster resonance energy transfer (FRET), up-conversion luminescence resonance energy transfer (ULRET), and photoelectrochemical (PEC) sensing. The drawbacks of MOF biosensors and the challenges encountered in the field are also briefly explored from our perspective.

Amyloid Typing in Cardiac Amyloidosis Using Western Blotting.

BACKGROUND: Cardiac amyloidosis (CA) is characterized by the extracellular deposition of misfolded protein in the heart. Precise identification of the amyloid type is often challenging, but critical, since the treatment and prognosis depend on the disease form and the type of deposited amyloid. Coexistence of clinical conditions such as old age, monoclonal gammopathy, chronic inflammation, or peripheral neuropathy in a patient with cardiomyopathy creates a differential diagnosis between the major types of CA: amyloidosis light chains (AL), amyloidosis transthyretin (ATTR) and amyloidosis A (AA). OBJECTIVES: To demonstrate the utility of the Western blotting (WB)-based amyloid typing method in patients diagnosed with cardiac amyloidosis where the type of amyloid was not obvious based on the clinical context. METHODS: Congo red positive endomyocardial biopsy specimens were studied in patients where the type of amyloid was uncertain. Amyloid proteins were extracted and identified by WB. Mass spectrometry (MS) of the electrophoretically resolved protein-in-gel bands was used for confirmation of WB data. RESULTS: WB analysis allowed differentiation between AL, AA, and ATTR in cardiac biopsies based on specific immunoreactivity of the electrophoretically separated proteins and their characteristic molecular weight. The obtained results were confirmed by MS. CONCLUSIONS: WB-based amyloid typing method is cheaper and more readily available than the complex and expensive gold standard techniques such as MS analysis or immunoelectron microscopy. Notably, it is more sensitive and specific than the commonly used immunohistochemical techniques and may provide an accessible diagnostic service to patients with amyloidosis in Israel.

Morphological findings in different subtypes of hepatic amyloid.

The classic findings have been well described for light-chain amyloid involving the liver. In addition to light chain, however, many additional proteins are now known to be amyloidogenic and can involve the liver. A total of 58 surgical pathology specimens with amyloid deposits were analyzed for patterns of amyloid deposition, including amyloid from light chain lambda (N = 17), light chain kappa (N = 15), transthyretin (N = 15), serum amyloid A (N = 4), apolipoprotein A1 (N = 4), fibrinogen alpha (N = 2), LECT2 (N = 1). Amyloid deposits predominately targeted the liver vasculature, including the walls of the hepatic arteries, portal veins, and sinusoids. While there was overlap, light chain amyloid predominately involved the sinusoids, while transthyretin amyloid predominately targeted the hepatic arteries, especially the larger ones in the hilum and larger portal tracts. Serum amyloid A formed nodular deposits that started in the portal vasculature but then extended into the portal tract stroma, leading to large, bulbous, portal-based amyloid deposits. Apolipoprotein A amyloid also formed large portal-based nodules. Fibrinogen was mild and subtle on H&E and predominately affected portal veins. Amyloid deposits in hilar nerves were prominent with amyloid light chain, transthyretin, and apolipoprotein A1. In conclusion, the histology of hepatic amyloid is diverse and shows several distinct clusters of findings that can aide in recognition in surgical pathology specimens.

Fine-needle aspiration of amyloidoma: A critical analysis.

BACKGROUND: Amyloid, presenting as a mass, is termed amyloidoma. Among the reported cases, fine-needle aspiration (FNA) of amyloid is often misinterpreted as acellular nondiagnostic material. METHODS: A computer search of all FNAs was performed and cases diagnosed as amyloidoma were identified. RESULTS: Among 11,956 cases and 20,634 FNAs, there were six cases and 12 FNAs of amyloidoma. One case with mucin/myxoid matrix was misinterpreted as amyloid, which on our review was Congo red negative. All five other cases of amyloidoma were adequate for evaluation. The smears showed most of the aspirated contents in the middle of the slide and it did not spread when smeared. The amyloid was present as large chunks of waxy, smooth, orangophilic/cyanophilic fragments on Papanicolaou stain and as basophilic fragments on Diff-Quik stain in a clean background. In cases with lymphoma/myeloma, there were admixed lymphocytes and/or plasma cells. Unlike fibrous tissue, amyloid aspirates well and provides adequate material for interpretation. The clean background distinguishes it from mucin/myxoid matrix. Congo red stain was positive with apple green birefringence in all five cases. Further subtyping by mass spectrometry showed AL (κ) type in three patients and AIns (insulin) type in one patient. In one patient with lymphoma, the subtyping was not done. CONCLUSION: FNA of amyloidoma is rare (0.04%), but an optimal method for diagnosis and subtyping, avoiding unwanted surgical interventions. Although mistaken for fibrous tissue, which aspirates poorly, abundant acellular orangophilic/cyanophilic material on FNA should raise a suspicion for amyloid. Unlike mucin/myxoid matrix, amyloid does not smear the background.

Oral manifestations of amyloidosis and the diagnostic applicability of oral tissue biopsy.

BACKGROUND: Amyloidosis exhibits a variable spectrum of systemic signs and oral manifestations that can be difficult to diagnose. This study aimed to characterize the clinical, demographic, and microscopic features of amyloidosis in the oral cavity. METHODS: This collaborative study involved three Brazilian oral pathology centers and described cases with a confirmed diagnosis of amyloidosis on available oral tissue biopsies. Clinical data were obtained from medical records. H&E, Congo-red, and immunohistochemically stained slides were analyzed. RESULTS: Twenty-six oral biopsies from 23 individuals (65.2% males; mean age: 59.6 years) were included. Oral involvement was the first sign of the disease in 67.0% of cases. Two patients had no clinical manifestation in the oral mucosa, although the histological analysis confirmed amyloid deposition. Amyloid deposits were distributed in perivascular (88.0%), periacinar and periductal (80.0%), perineurial (80.0%), endoneurial (33.3%), perimuscular (88.2%), intramuscular (94.1%), and subepithelial (35.3%) sites as well as around fat cells (100.0%). Mild/moderate inflammation was found in 65.4% of cases and 23.1% had giant cells. CONCLUSIONS: Amyloid deposits were consistently found in oral tissues, exhibiting distinct deposition patterns. Oral biopsy is less invasive than internal organ biopsy and enables the reliable identification of amyloid deposits even in the absence of oral manifestations. These findings corroborate the relevance of oral biopsy for the diagnosis of amyloidosis.

Hyperspectral Raman Imaging for Automated Recognition of Human Renal Amyloid.

In the clinical setting, routine identification of the main types of tissue amyloid deposits, light-chain amyloid (AL) and serum amyloid A (AA), is based on histochemical staining; rarer types of amyloid require mass spectrometry analysis. Raman spectroscopic imaging is an analytical tool, which can be used to chemically map, and thus characterize, the molecular composition of fluid and solid tissue. In this proof-of-concept study, we tested the feasibility of applying Raman spectroscopy combined with artificial intelligence to detect and characterize amyloid deposits in unstained frozen tissue sections from kidney biopsies with pathologic diagnosis of AL and AA amyloidosis and control biopsies with no amyloidosis (NA). Raman hyperspectral images, mapped in a 2D grid-like fashion over the tissue sections, were obtained. Three machine learning-assisted analysis models of the hyperspectral images could accurately distinguish AL (types λ and κ), AA, and NA 93-100% of the time. Although very preliminary, these findings illustrate the potential of Raman spectroscopy as a technique to identify, and possibly, subtype renal amyloidosis.

Diabetes mellitus drug discovery: insights into targeting feline and human amylin with small molecules.

BACKGROUND: Type 2 diabetes (T2D) is a health concern for both humans and cats, with cases rising over the past decade. Around 70% of patients from either species exhibit pancreatic aggregates of islet amyloid polypeptide (IAPP), a protein that proves toxic upon misfolding. These misfolded protein aggregates congregate in the islets of Langerhans of the pancreas, diminishing the capability of ß-cells to produce insulin and further perpetuating disease. OBJECTIVE: Our team's drug discovery program is investigating newly synthesized compounds that could diminish aggregates of both human and feline IAPP, potentially disrupting the progression of T2D. MATERIAL AND METHODS: We prepared 24 compounds derived from diaryl urea, as ureas have previously demonstrated great potential at reducing accumulations of misfolded proteins. Biophysical methods were employed to analyze the anti-aggregation activity of these compounds at inhibiting and/or disrupting IAPP fibril formation in vitro. RESULTS: The results demonstrate that compounds 12 and 24 were most effective at reducing the fibrillization and aggregation of both human and feline IAPP. When compared with the control for each experiment, samples treated with either compound 12 or 24 exhibited fewer accumulations of amyloid-like fibrils. CONCLUSION: Urea-based compounds, such as compounds 12 and 24, may prove crucial in future pre-clinical studies in the search for therapeutics for T2D.

Proteomic and Clinicopathologic Assessment of Penile Amyloidosis: A Single Institutional Review of 12 Cases.

OBJECTIVES: There is a paucity of data on penile amyloidosis. We aimed to assess the frequency of different amyloid types in surgical specimens from the penis involved by amyloidosis and correlate relevant clinicopathologic parameters with proteomic findings. METHODS: Since 2008, our reference laboratory has performed liquid chromatography/tandem mass spectrometry (LC-MS/MS) for amyloid typing. The institutional pathology archive and reference laboratory database were queried to retrospectively identify all penile surgical pathology specimens with LC-MS/MS results between January 1, 2008, and November 23, 2022. Archived H&E-stained and Congo red-stained sections were re-reviewed. RESULTS: Twelve cases of penile amyloidosis were identified, which represented 0.35% (n = 3,456) of penile surgical specimens. AL-type amyloid was most frequent (n = 7), followed by keratin-type amyloid (n = 3) and ATTR (transthyretin)-type amyloid (n = 2). AL-type amyloid cases often showed diffuse dermal/lamina propria deposition, whereas all keratin-type amyloid cases were localized to the superficial dermis. Two cases with keratin-type amyloid had concomitant cutaneous findings (penile intraepithelial neoplasia and condyloma). CONCLUSIONS: This series, the largest to date, demonstrates that penile amyloidosis has a heterogeneous proteomic landscape. To the best of our knowledge, this is the first study describing ATTR (transthyretin)-type penile amyloid.

Putative Structures of Membrane-Embedded Amyloid ß Oligomers.

Perturbation of cell membranes by amyloid ß (Ab) peptide oligomers is one possible mechanism of cytotoxicity in Alzheimer's disease, but the structure of such Ab-membrane complexes is unknown. Here we examine the stability of several putative structures by implicit membrane and all-atom molecular dynamics simulations. The structures include (a) a variety of models proposed by other researchers in the past, (b) a heptameric ß barrel determined by grafting the Ab sequence onto α-hemolysin, (c) a similar structure with modified strand orientation and turn location based on an experimental ß-hairpin structure, (d) oligomers inserting C-terminal ß hairpins into one leaflet of the bilayer, (e) oligomers forming parallel C-terminal ß barrels, and (f) a helical hexamer made of C-terminal fragments. The α-hemolysin-grafted structure and its alternately oriented variant are stable in the membrane and form an aqueous pore. In contrast, the C-terminal parallel barrels are not stable, presumably due to excessive hydrophobicity of their inner surface. The helical hexamer also failed to stabilize an aqueous pore for the same reason. The C-terminal hairpin-inserting structures remain stably inserted but, again, do not form an aqueous pore. Our results suggest that only ß-barrels inserting a combination of C-terminal and other residues can form stable aqueous pores.

Spectral Phasor Analysis of Nile Red Identifies Membrane Microenvironment Changes in the Presence of Amyloid Peptides.

The interaction of protein and peptide amyloid oligomers with membranes is thought to be one of the mechanisms contributing to cellular toxicity. However, techniques to study these interactions in the complex membrane environment of live cells are lacking. Spectral phasor analysis is a recently developed biophysical technique that can enable visualisation and analysis of membrane-associated fluorescent dyes. When the spectral profile of these dyes changes as a result of changes to the membrane microenvironment, spectral phasor analysis can localise those changes to discrete membrane regions. In this study, we investigated whether spectral phasor analysis could detect changes in the membrane microenvironment of live cells in the presence of fibrillar aggregates of the disease-related Aß42 peptide or the functional amyloid neurokinin B. Our results show that the fibrils cause distinct changes to the microenvironment of nile red associated with both the plasma and the nuclear membrane. We attribute these shifts in nile red spectral properties to changes in membrane fluidity. Results from this work suggest that cells have mechanisms to avoid or control membrane interactions arising from functional amyloids which have implications for how these peptides are stored in dense core vesicles. Furthermore, the work highlights the utility of spectral phasor analysis to monitor microenvironment changes to fluorescent probes in live cells.

Death occurred due to undiagnosed systemic amyloidosis: a case report.

Abstract: Amyloidosis is a disorder related to errors in protein folding. We present a clinical case of systemic amyloidosis manifesting as hypotension, tachycardia, pain, weight loss, asthenia, anorexia, dysphagia, and mood deflection in a 49-year-old-year-old woman with a previous clinical history of articular and muscular pain, correlated to suspected seronegative arthritis. The blood test revealed kidney insufficiency, an electrocardiogram identified low voltages of the peripheral leads and T waves anomalies. A serum protein electrophoresis revealed the presence of high levels of monoclonal kappa free chains. The woman started to have a sense of suffocation, and after one week she was found dead in her bed. After the autopsy, the results of Congo red staining of the myocardium were characteristic of amyloid. According to the autoptic and the histological examination, death occurred due to acute cardiac and respiratory arrest secondary to amyloid cardiomyopathy in a patient with undiagnosed systemic amyloidosis.

[Transthyretin amyloid cardiomyopathy. Features of histological diagnosis: study design].

AIM: To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes. MATERIALS AND METHODS: Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet. RESULTS: Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils. CONCLUSION: The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.

Atomic Insights into Amyloid-Induced Membrane Damage.

Amphipathic peptides can cause biological membranes to leak either by dissolving their lipid content via a detergent-like mechanism or by forming pores on the membrane surface. These modes of membrane damage have been related to the toxicity of amyloid peptides and to the activity of antimicrobial peptides. Here, we perform the first all-atom simulations in which membrane-bound amphipathic peptides self-assemble into ß-sheets that subsequently either form stable pores inside the bilayer or drag lipids out of the membrane surface. An analysis of these simulations shows that the acyl tail of lipids interact strongly with non-polar side chains of peptides deposited on the membrane. These strong interactions enable lipids to be dragged out of the bilayer by oligomeric structures accounting for detergent-like damage. They also disturb the orientation of lipid tails in the vicinity of peptides. These distortions are minimized around pore structures. We also show that membrane-bound ß-sheets become twisted with one of their extremities partially penetrating the lipid bilayer. This allows peptides on opposite leaflets to interact and form a long transmembrane ß-sheet, which initiates poration. In simulations, where peptides are deposited on a single leaflet, the twist in ß-sheets allows them to penetrate the membrane and form pores. In addition, our simulations show that fibril-like structures produce little damage to lipid membranes, as non-polar side chains in these structures are unavailable to interact with the acyl tail of lipids.

Cutaneous Ossifying Amyloidoma.

ABSTRACT: Amyloidoma, otherwise known as tumoral amyloidosis, is a localized deposition of amyloid (AL-type or AA type) without systemic amyloidosis. It is the rarest form of tissue amyloid deposition, and up to 7% of amyloidomas develop systemic amyloidosis.Cutaneous AL-type amyloidoma is considered by many authors as an unusual variant of primary cutaneous marginal zone lymphoma. Although cutaneous amyloidoma can form calcifications, ossification is extremely unusual, with only 1 case previously published to date.We report the case of a 75-year-old woman with voluminous and strikingly ossifying AL-type amyloidoma in the left pretibial skin. Her medical history included excision of hepatic hydatidic cysts 25 years prior and diffuse large B-cell lymphoma of the left parotid gland 8 years prior treated with chemotherapy and radiotherapy with complete response. After the diagnosis of amyloidoma, an extension study with cervical, chest, abdominal, and pelvic TC was performed, with no additional lesions found. Serum and protein electrophoresis revealed elevations in kappa light chain and IgA immunoglobulin levels but did not reveal monoclonal bands. In situ hybridization for immunoglobulin light chains showed monotypic kappa expression in plasma cells infiltrating the amyloidoma.Extensive ossification in amyloidomas can make diagnosis difficult; therefore, we describe an interesting case of this histopathologically peculiar amyloidoma.

Analysis of lumbar spine stenosis specimens for identification of amyloid.

BACKGROUND: Lumbar spinal stenosis (LSS) is a common reason for spine surgery in which ligamentum flavum is resected. Transthyretin (TTR) amyloid is an often unrecognized and potentially modifiable mechanism for LSS that can also cause TTR cardiac amyloidosis. Accordingly, older adult patients undergoing lumbar spine (LS) surgery were evaluated for amyloid and if present, the precursor protein, as well as comprehensive characterization of the clinical phenotype. METHODS: A prospective, cohort study in 2 academic medical centers enrolled 47 subjects (age 69 ± 7 years, 53% male) undergoing clinically indicated LS decompression. The presence of amyloid was evaluated by Congo Red staining and in those with amyloid, precursor protein was determined by laser capture microdissection coupled to mass spectrometry (LCM-MS). The phenotype was assessed by disease-specific questionnaires (Swiss Spinal Stenosis Questionnaire and Kansas City Cardiomyopathy Questionnaire) and the 36-question short-form health survey, as well as biochemical measures (TTR, retinol-binding protein, and TTR stability). Cardiac testing included technetium-99m-pyrophosphate scintigraphy, electrocardiograms, echocardiograms, and cardiac biomarkers as well as measures of functional capacity. RESULTS: Amyloid was detected in 16 samples (34% of participants) and was more common in those aged ≥ 75 years of age (66.7%) compared with those <75 years (22.3%, p < 0.05). LCM-MS demonstrated TTR as the precursor protein in 62.5% of participants with amyloid while 37.5% had an indeterminant type of amyloid. Demographic, clinical, quality-of-life measures, electrocardiographic, echocardiographic, and biochemical measures did not differ between those with and without amyloid. Among those with TTR amyloid (n = 10), one subject had cardiac involvement by scintigraphy. CONCLUSIONS: Amyloid is detected in more than a third of older adults undergoing LSS. Amyloid is more common with advancing age and is particularly common in those >75 years old. No demographic, clinical, biochemical, or cardiac parameter distinguished those with and without amyloid. In more than half of subjects with LS amyloid, the precursor protein was TTR indicating the importance of pathological assessment.

Tandem Mass Spectrometry-Based Amyloid Typing Using Manual Microdissection and Open-Source Data Processing.

OBJECTIVES: Standard implementations of amyloid typing by liquid chromatography-tandem mass spectrometry use capabilities unavailable to most clinical laboratories. To improve accessibility of this testing, we explored easier approaches to tissue sampling and data processing. METHODS: We validated a typing method using manual sampling in place of laser microdissection, pairing the technique with a semiquantitative measure of sampling adequacy. In addition, we created an open-source data processing workflow (Crux Pipeline) for clinical users. RESULTS: Cases of amyloidosis spanning the major types were distinguishable with 100% specificity using measurements of individual amyloidogenic proteins or in combination with the ratio of λ and κ constant regions. Crux Pipeline allowed for rapid, batched data processing, integrating the steps of peptide identification, statistical confidence estimation, and label-free protein quantification. CONCLUSIONS: Accurate mass spectrometry-based amyloid typing is possible without laser microdissection. To facilitate entry into solid tissue proteomics, newcomers can leverage manual sampling approaches in combination with Crux Pipeline and related tools.

Amyloid fibril formation by casein and fatty acid composition in breast milk of mastitis patients.

Mastitis can cause changes in the nutrient composition of breast milk, which may be harmful to both newborns and lactating mothers. In this study we preliminarily evaluated amyloid fibrils formation by casein and fatty acids (FA), as well as their potential relation with each other in the breast milk of mastitis patients. Six healthy volunteers and six mastitis patients were recruited from the Maternal and Child Health Care Hospital in Changchun were enrolled. Amyloid fibril content was assessed by thioflavin T fluorescence analysis, transmission electron microscope, circular dichroism, and proton nuclear magnetic resonance. FA contents were measured by gas chromatography. Healthy breast milk contained no amyloid fibrils but inflammatory breast milk did. Several FAs (hendecanoic acid, myristolenic acid, pentadecenoic acid, eicosatrienoic acid) differed significantly between the two groups (p < .05). The concentrations of the eicosatrienoic acid and eleven carbonic acids in the inflammatory groups were lower than those in the healthy groups, but the myristolenic acid and pentadecenoic acid were the opposite trend. Early detection of amyloid fibrils should be performed in lactating mothers with mastitis. Changes in FAs may reflect the importance of abnormal metabolism in amyloid fibril formation. PRACTICAL APPLICATIONS: The work preliminarily clarified the relationship between inflammation, fibril content, and fatty acid (FA) composition in breast milk. Healthy milk contained no amyloid fibril formed by casein but the inflammatory milk did. FAs were also significantly different between the two groups. Thus, an early determination of amyloid fibrils in milk should be considered for lactating women with mastitis to avoid the further malignant development. Additionally, the changes in FAs may reflect the importance of abnormal metabolism and oxidative pathways in amyloid fibril formation in the breast. Therefore, this study provided foundations for further investigation on the association between inflammation, fibril content and FA composition in breast milk.

A case of medical liability involving an unexpected systemic amyloidosis.

The authors present a case of fatal amyloid cardiomyopathy, which was diagnosed only upon autopsy. A 57-year-old man was admitted to the hospital for scheduled percutaneous cardiac procedure of transcatheter radiofrequency ablation due to persistent atrial fibrillation and atrial flutter. Ventricular fibrillation was recorded in the monitor 2 h after the surgical procedure. Therefore, he was defibrillated and intubated, but he died for nosocomial pneumonia 26 days after being admitted. A judicial autopsy was ordered by the prosecutor due to an alleged medical malpractice. The autopsy confirmed the cause of death being pneumonia, but also revealed an occult restrictive cardiomyopathy with a thick and firm myocardium. Viscera samples were then collected for microscopic examination. Histopathologic analysis showed diffuse amyloid deposits in the myocardium, especially in the perivascular and subendocardial spaces. Amyloid deposits were also detected in all the other organs, except for the brain. Furthermore, immunohistochemistry for light chains was performed on the heart tissue sample, resulting to be positive. In the case presented herein, autopsy and histopathologic examination were crucial to diagnose an occult systemic amyloidosis (AL-type). In fact, it has been observed that the rarity of systematic amyloidosis and its unusual clinical onset were at first mistakenly perceived as a medical malpractice due to a technical error within the catheter ablation for atrial fibrillation. As a consequence, upon discussing the clinical and medicolegal implications concerning the case, the focus was placed on the undiagnosed systemic amyloidosis and on the causality between surgical procedure and the patient's death.

Bone marrow amyloid: a comprehensive analysis of 1,469 samples, including amyloid type, clinical features, and morphologic distribution.

BACKGROUND: Bone marrow biopsy is common in patients suspected of having systemic AL amyloidosis. However, little is known about the incidence, morphology and clinical phenotype of non-AL amyloid types in bone marrow. METHODS: We retrospectively identified N = 1469 bone marrow amyloid biopsies typed using a proteomics-based method between 2008-2020. Frequency of amyloid types (N = 1469), distribution of amyloid deposits (N = 139), and clinical phenotypes (N = 355), with particular emphasis on cardiac involvement, were assessed. RESULTS: The amyloid types were: AL (N = 1172; 79.8%), ATTR (N = 240; 16.3%), AH (N = 38; 2.6%), AA (N = 17; 1.2%), and Aß2M (N = 2; 0.1%). Although there were characteristic morphologic features, including periosteal soft tissue and/or vascular involvement in ATTR, interstitial vascular involvement in AA, and variable anatomic compartment involvement in AL, none were pathognomonic. Most patients with both an M-spike and cardiac involvement had AL amyloid in their BM, but in over 10% the amyloid type was ATTR. Compared to AL patients, ATTR patients had higher stage cardiac amyloidosis and lower overall survival, which was mainly due to advanced cardiac stage. CONCLUSIONS: ATTR amyloid is common in bone marrow and its morphologic distribution overlaps with AL. Amyloid typing is critical as over 10% of patients with bone marrow amyloid, cardiac amyloidosis, and an M-spike have ATTR amyloidosis.