Quality assessment of histopathological stainings on prolonged formalin fixed thrombus tissues retrieved by mechanical thrombectomy.

Background: Formalin-fixed retrieved clots from mechanical thrombectomy (MT) are now routinely studied using both conventional histopathologic techniques and immunohistochemistry (IHC). However, the effects of prolonged formalin fixation on the histological results of clot analysis remain unknown. The objective of this study was to investigate the effects of prolonged formalin fixation on quality of histopathologic stainings of thrombus tissues retrieved by MT. Methods: As part of the multicenter EXCELLENT registry, a total of 80 clots extracted by MT from acute ischemic stroke patients were randomly selected from the tissue database and assigned into four groups according to 10% neutral buffered formalin (NBF) fixation duration (1-30, 30-60, 60-90, and 90+ days, up to 2 years). Samples underwent processing and sectioning. Two serial sections for each case were stained with hematoxylin and eosin (H&E), Martius Scarlet Blue (MSB), and IHC for CD42b (platelet marker). An expert pathologist, who was blinded to tissue fixation duration and patient clinical data, assessed the quality of each stain including stainability, sensitivity, specificity, and consistency of stainings. Results: No significant issues were encountered during tissue processing and sectioning. On H&E stain, 97.5% (78/80) of slides showed good-quality staining, demonstrating clear histological properties of the thrombus tissue as red blood cells (RBC) stained in red, fibrin/platelet stained in pink, and nuclei stained in blue with intranuclear detail. The same histological features were also successfully demonstrated on MSB for all 80 samples. One of the 80 samples (1.2%) showed that RBC lost stainability on H&E due to tissue autolysis. Clear positive signal of platelet staining was expressed in 98.8% of the samples (79/80) with minimal background staining on IHC. There was no significant difference in staining quality across different formalin fixation groups. Conclusion: A good quality of histopathological staining is achievable for the thrombus tissue fixed in 10% neutral buffered formalin for up to 2 years. The findings are limited to the thrombus tissue retrieved by MT and specific fixation and staining protocols used in the study. To apply these results to other tissue or experimental setups, further studies and validations would be necessary. Clinical trial registration: This study was conducted as part of the EXCELLENT study: www.clinicaltrials.gov, unique identifier: NCT03685578.

Correlation between acute ischaemic stroke clot length before mechanical thrombectomy and extracted clot area: Impact of thrombus size on number of passes for clot removal and final recanalization.

INTRODUCTION: We assessed the correlation between thrombus size before and after mechanical thrombectomy, measured as length by Computed Tomography Angiography/Non-Contrast Computed Tomography (CTA/NCCT) and Extracted Clot Area, ECA, respectively. We also assessed the influence of thrombus size on the number of passes required for clot removal and final recanalization outcome. MATERIALS AND METHODS: Acute ischaemic stroke (AIS) thrombi retrieved by mechanical thrombectomy from 500 patients and data of clot length by CTA/NCCT were collected from three hospitals in Europe. ECA was obtained by measuring the area of the extracted clot. Non-parametric tests were used for data analysis. RESULTS: A strong positive correlation was found between clot length on CTA/NCCT and ECA (rho = 0.619,N = 500,P < 0.0001*). Vessel size influences clot length on CTA/NCCT (H2 = 98.6, P < 0.0001*) and ECA (H2 = 105.6,P < 0.0001*), but the significant correlation between CTA/NCCT length and ECA was evident in all vessels. Poorer revascularisation outcome was associated with more passes (H5 = 73.1, P < 0.0001*). More passes were required to remove longer clots (CTA/NCCT; H4 = 31.4, P < 0.0001*; ECA; H4 = 50.2, P < 0.0001*). There was no significant main association between recanalization outcome and length on CTA/NCCT or ECA, but medium sized clots (ECA 20-40 mm2) were associated with least passes and highest revascularisation outcome (N = 500, X2 = 16.2, P < 0.0001*). CONCLUSION: Clot length on CTA/NCCT strongly correlates with ECA. Occlusion location influences clot size. More passes are associated with poorer revascularisation outcome and bigger clots. The relationship between size and revascularisation outcome is more complex. Clots of medium ECA take less passes to remove and are associated with better recanalization outcome than both smaller and larger clots.

Per pass analysis of thrombus composition retrieved by mechanical thrombectomy.

BACKGROUND AND AIM: Mechanical thrombectomy (MT) for large vessel occlusion often requires multiple passes to retrieve the entire thrombus load. In this multi-institutional study we sought to examine the composition of thrombus fragments retrieved with each pass during MT. METHODS: Patients who required multiple passes during thrombectomy were included. Histopathological evaluation of thrombus fragments retrieved from each pass was performed using Martius Scarlet Blue staining and the composition of each thrombus component including RBC, fibrin and platelet was determined using image analysis software. RESULTS: 154 patients underwent MT and 868 passes was performed which resulted in 263 thrombus fragments retrieval. The analysis of thrombus components per pass showed higher RBC, lower fibrin and platelet composition in the pass 1 and 2 when compared to pass 3 and passes 4 or more combined (P values <0.05). There were no significant differences between thrombus fragments retrieved in pass 1 and pass 2 in terms of RBC, WBC, fibrin, and platelet composition (P values >0.05). Similarly, when each composition of thrombus fragments retrieved in pass 3 and passes 4 or more combined were compared with each other, no significant difference was noted (P values >0.05). CONCLUSION: Our findings confirm that thrombus fragments retrieved with each pass differed significantly in histological content. Fragments in the first passes were associated with lower fibrin and platelet composition compared to fragments retrieved in passes three and four or higher. Also, thrombus fragments retrieved after failed pass were associated with higher fibrin and platelet components.

Correlation of imaging and histopathology of thrombi in acute ischemic stroke with etiology and outcome.

Mechanical thrombectomy has become the stand of care for patients with large vessel occlusions, yet major improvements in thrombectomy speed, efficacy, and completeness can still be achieved. High rates of clot fragmentation and failure to remove the clot resulting in poor neurological outcomes suggest that in order to further advance the field of stroke intervention we must turn our attention towards understanding the science of clot. Accurately identifying the composition of the occlusive clot prior to intervention could significantly influence the success of the revascularization strategy used to treat them. Numerous features of thromboemboli could be studied and characterized, including quantitative histomorphometry and diagnostic imaging characteristics. Each of these features might logically predict superior thrombectomy outcomes with one device or another. This article aims to review the current literature on histopathological composition of acute ischemic stroke clots, with a particular focus on the correlation between clot composition and diagnostic imaging, stroke etiology and revascularization outcomes.

Correlation of histomorphological pattern of cardiac amyloid deposition with amyloid type: a histological and proteomic analysis of 108 cases.

AIMS: Prognostication and treatment selection for cardiac amyloidosis requires accurate amyloid typing. The aim of this study was to investigate the utility of histomorphology for predicting type. METHODS AND RESULTS: Autopsy cases with cardiac amyloidosis (1998-2010) were typed by the use of mass spectrometry-based proteomics. Deposition patterns were correlated with amyloid type. Among 108 decedents (mean age 75 years; 69% men), 107 had a single type, including transthyretin (ATTR) (60 cases), amyloid light chain (AL) (32 λ; 12 κ), amyloid A (AA) (two), and apolipoprotein AIV (AApoAIV) (one). Interstitial deposition was more extensive in AL amyloidosis cases than in ATTR cases [odds ratio (OR) 6.8, P = 0.0004]. Histomorphological patterns of interstitial deposition were mixed in 61% of AL amyloidosis cases and in 61% of ATTR cases, but diffuse pericellular deposits favoured AL amyloidosis (OR 10.7, P = 0.0001), nodular deposits favoured ATTR (OR 3.1, P = 0.0229), and discrete pericellular deposits tended to partially favour ATTR (OR 1.7, P = 0.1970). Arterial and venous deposits each favoured AL amyloidosis (OR ranging from 9.3 to 192.0, P-value ranging from 0.0022 to <0.0001), and were severe in AL amyloidosis. Endocardial deposits favoured AL amyloidosis (OR 46.3, P < 0.0001) and were also more severe in AL amyloidosis. CONCLUSIONS: The extent and distribution of cardiac amyloidosis strongly correlate with amyloid type, suggesting fundamental differences in the pathobiology of deposition. The tendency for mixed patterns to occur limits the practicality and accuracy of using histopathology for amyloid typing.

Globular hepatic amyloid is highly sensitive and specific for LECT2 amyloidosis.

Globular hepatic amyloid (GHA) is rare, and its clinical significance remains unclear. Recently, leukocyte chemotactic factor-associated amyloidosis (ALECT2) has been reported to involve the liver, showing a globular pattern. We reviewed 70 consecutive cases of hepatic amyloidosis to determine the prevalence and morphology of hepatic amyloid subtypes, especially ALECT2 and its association with GHA. Each case was reviewed for amyloid subtype (immunohistochemistry and/or mass spectrometry), its pattern (linear or globular), and distribution (vascular, perisinusoidal, or stromal). In addition, 24 cases of confirmed hepatic ALECT2 on mass spectrometry from our consultation files were also reviewed. LECT2 immunostaining was performed in 49 cases. Of the 70 cases, immunoglobulin light chain (AL) type was most common with 41 cases (59%), followed by transthyretin (ATTR) 15 cases (22%), 3 cases each of fibrinogen A (AFib) (4%), serum amyloid A (AA) (4%), and ALECT2 (4%), 2 cases of apolipoproteins (AApoA1) (3%), and 3 cases (4%) were unclassified. Three of our 70 cases (4%), with ALECT2, and all 24 cases (100%) of mass spectrometry-confirmed hepatic ALECT2 showed only GHA deposits in the hepatic sinusoids and portal tracts. Three (4%) other cases of AL type showed a focal globular pattern admixed with prominent linear amyloid. None of the other amyloid subtypes showed GHA. LECT2 immunostain was positive in all 27 cases (100%) of ALECT2 and negative in the other 22 non-ALECT2 cases (100%) (14 AL, 5 ATTR, 1 AA, 1 AFib, 1 AApoA1). Pure GHA is uncommon (4%) but is highly specific for ALECT2, and LECT2 immunostain is helpful in confirming this amyloid type.

Impact of incidental amyloidosis on the prognosis of patients with hypertrophic cardiomyopathy undergoing septal myectomy for left ventricular outflow tract obstruction.

To determine the impact of amyloid on the prognosis of patients with hypertrophic cardiomyopathy (HC), we reviewed outcomes of patients who underwent septal myectomy for HC from March 7, 1996, to October 9, 2012, with amyloid deposits identified in operative specimens. Amyloid subtypes were differentiated by mass spectrometry-based proteomics. The survival rate was compared with that of an age-matched population (2:1) without amyloid who underwent septal myectomy for HC. Sixteen patients (mean age ± SD 71 ± 8 years; 12 men) met study criteria. All 16 had intraventricular peak systolic gradients reduced intraoperatively from 105 ± 53 mm Hg to 3 ± 7 mm Hg (p <0.001). Amyloid deposits in specimens ranged from minimal to mild. Nine patients had senile (transthyretin-type) amyloidosis, 4 had immunoglobulin-associated amyloidosis, 2 had apolipoprotein A4 amyloidosis type, and 1 had serum amyloid A type. There were no deaths before 30 days. Twelve patients had New York Heart Association class III or IV function preoperatively, and at last follow-up (median 3 years), class I or II. Only 1 patient received postoperative amyloidosis treatment. The postoperative survival rate at 2 and 4 years was 100% (n = 11 at risk) and 91% (n = 6 at risk), respectively, similar to that of the age-matched population with HC without amyloid who underwent myectomy (p = 0.13). Patients undergoing septal myectomy for HC who have histologic evidence of mild amyloidosis have early outcomes and midterm survival similar to those of patients with HC without amyloidosis who undergo myectomy. In conclusion, although longer follow-up is necessary, small amounts of amyloid, regardless of subtype, do not confer a poor prognosis on patients with HC who undergo septal myectomy.

Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics.

Examination of abdominal subcutaneous fat aspirates is a practical, sensitive and specific method for the diagnosis of systemic amyloidosis. Here we describe the development and implementation of a clinical assay using mass spectrometry-based proteomics to type amyloidosis in subcutaneous fat aspirates. First, we validated the assay comparing amyloid-positive (n=43) and -negative (n=26) subcutaneous fat aspirates. The assay classified amyloidosis with 88% sensitivity and 96% specificity. We then implemented the assay as a clinical test, and analyzed 366 amyloid-positive subcutaneous fat aspirates in a 4-year period as part of routine clinical care. The assay had a sensitivity of 90%, and diverse amyloid types, including immunoglobulin light chain (74%), transthyretin (13%), serum amyloid A (%1), gelsolin (1%), and lysozyme (1%), were identified. Using bioinformatics, we identified a universal amyloid proteome signature, which has high sensitivity and specificity for amyloidosis similar to that of Congo red staining. We curated proteome databases which included variant proteins associated with systemic amyloidosis, and identified clonotypic immunoglobulin variable gene usage in immunoglobulin light chain amyloidosis, and the variant peptides in hereditary transthyretin amyloidosis. In conclusion, mass spectrometry-based proteomic analysis of subcutaneous fat aspirates offers a powerful tool for the diagnosis and typing of systemic amyloidosis. The assay reveals the underlying pathogenesis by identifying variable gene usage in immunoglobulin light chains and the variant peptides in hereditary amyloidosis.

Leukocyte cell-derived chemotaxin 2 (LECT2)-associated amyloidosis is a frequent cause of hepatic amyloidosis in the United States.

Using laser microdissection and mass spectrometry (MS)-based proteomics, we subtyped amyloid deposits from 130 cases of hepatic amyloidosis. Although we confirmed that immunoglobulin light chain amyloidosis was the most frequent cause of hepatic amyloidosis, leukocyte cell-derived chemotaxin 2 (LECT2) amyloidosis (ALect2) accounted for 25% of cases. This novel finding was associated with Hispanic ancestry, incidental discovery of amyloid in liver specimens sampled for other unrelated conditions, and a characteristic pattern of hepatic amyloid deposition. Although ALect2 patients had a common LECT2 polymorphism, pathogenic mutations were not discovered, suggesting that constitutive or compensatory LECT2 overexpression led to ALect2 deposition. These findings indicate that ALect2 is a common cause of hepatic amyloidosis in the population of the United States, and subtyping hepatic amyloid deposits by an accurate analytic method such as MS is required for optimal clinical management of hepatic amyloidosis patients and to avoid incorrect and unnecessarily toxic therapies.

Systemic AA amyloidosis as a unique manifestation of a combined mutation of TNFRSF1A and MEFV genes.

We report the case of a 22-year-old Caucasian woman presenting with a new-onset nephrotic syndrome with normal renal function during the 35th week of pregnancy. AA (secondary) amyloidosis was further diagnosed at the renal biopsy. Extensive genetic testing revealed that the patient was heterozygous for both TNFRSF1A p.R92Q and MEFV p.M694I mutations leading to an autoinflammatory syndrome characterized by amyloid deposition as the sole manifestation.

Interactions between osteosarcoma cell lines and dendritic cells immune function: An in vitro study.

Dendritic cells (DCs) might be partly responsible for the defective immune response in tumor bearing hosts, but no study in osteosarcoma patients is still available. Therefore, we investigated in vitro whether human osteosarcoma cell lines have an inhibitor effect on different types of DCs: CD14+DCs, DC1 and DC2. DCs derived from healthy donors were cultured with osteosarcoma cell lines and appropriate cytokine cocktails and analysed for the expression of co-stimulatory molecules (CD40, CD80, CD83, CD86, HLA-DR). Each interaction resulted in a lower phenotypic expression of the DCs maturation markers, especially on DC2. Moreover, the addition of various cytokines and compounds (rhIL-12, CD40L, Indometacin) induced the DC1 and DC2 subsets towards the Th1 pattern as shown by ELISA. Osteosarcoma highly interferes with an in vitro DCs immune function as antigen presenting cells. The understanding of tumor biology underlines the need for a specific osteosarcoma immunotherapy able to reverse this immune-surveillance inhibition.