Clinical course and follow-up of pediatric patients with COVID-19 vaccine-associated myocarditis compared to non-vaccine-associated myocarditis within the prospective multicenter registry-"MYKKE".

BACKGROUND: Since the onset of widespread COVID-19 vaccination, increased incidence of COVID-19 vaccine-associated myocarditis (VA-myocarditis) has been noted, particularly in male adolescents. METHODS: Patients <18 years with suspected myocarditis following COVID-19 vaccination within 21 days were enrolled in the PedMYCVAC cohort, a substudy within the prospective multicenter registry for pediatric myocarditis "MYKKE." Clinical data at initial admission, 3- and 9-months follow-up were monitored and compared to pediatric patients with confirmed non-vaccine-associated myocarditis (NVA-myocarditis) adjusting for various baseline characteristics. RESULTS: From July 2021 to December 2022, 56 patients with VA-myocarditis across 15 centers were enrolled (median age 16.3 years, 91% male). Initially, 11 patients (20%) had mildly reduced left ventricular ejection fraction (LVEF; 45%-54%). No incidents of severe heart failure, transplantation or death were observed. Of 49 patients at 3-months follow-up (median (IQR) 94 (63-118) days), residual symptoms were registered in 14 patients (29%), most commonly atypical intermittent chest pain and fatigue. Diagnostic abnormalities remained in 23 patients (47%). Of 21 patients at 9-months follow-up (259 (218-319) days), all were free of symptoms and diagnostic abnormalities remained in 9 patients (43%). These residuals were mostly residual late gadolinium enhancement in magnetic resonance imaging. Patients with NVA-myocarditis (n=108) more often had symptoms of heart failure (P = .003), arrhythmias (P = .031), left ventricular dilatation (P = .045), lower LVEF (P < .001) and major cardiac adverse events (P = .102). CONCLUSIONS: Course of COVID-19 vaccine-associated myocarditis in pediatric patients seems to be mild and differs from non-vaccine-associated myocarditis. Due to a considerable number of residual symptoms and diagnostic abnormalities at follow-up, further studies are needed to define its long-term implications.

Screening for potential targets to reduce stenosis in bioprosthetic heart valves.

Progressive stenosis is one of the main factors that limit the lifetime of bioprosthetic valved conduits. To improve long-term performance we aimed to identify targets that inhibit pannus formation on conduit walls. From 11 explanted, obstructed, RNAlater presevered pulmonary valved conduits, we dissected the thickened conduit wall and the thin leaflet to determine gene expression-profiles using ultra deep sequencing. Differential gene expression between pannus and leaflet provided the dataset that was screened for potential targets. Promising target candidates were immunohistologically stained to see protein abundance and the expressing cell type(s). While immunostainings for DDR2 and FGFR2 remained inconclusive, EGFR, ErbB4 and FLT4 were specifically expressed in a subset of tissue macrophages, a cell type known to regulate the initiation, maintenance, and resolution of tissue repair. Taken toghether, our data suggest EGFR, ErbB4 and FLT4 as potential target candidates to limit pannus formation in bioprosthestic replacement valves.

ADAPT-treated pericardium for aortic valve reconstruction in congenital heart disease: histological analysis of a series of human explants.

OBJECTIVES: Different types of patch materials are used for aortic valve repair in children with congenital aortic valve disease to avoid early valve replacement. CardioCel© (Admedus, Toowong, QLD, Australia) consists of bovine pericardium treated with the ADAPT method (Admedus' proprietary tissue engineering process). METHODS: Our goal was to describe tissue reactions in 12 explanted aortic valve leaflet specimens (augmented or replaced with CardioCel patch material) (11 explanted surgically, 1 autopsy). Explantation was performed during reoperation after aortic valve repair, necessitated by aortic valve stenosis in 5, aortic valve insufficiency in 2, combined aortic valve lesions in 3 patients and endocarditis in 1 patient. One patient died of sudden left heart failure 28 months after aortic valve repair. At the last documented follow-up of this patient at 22 months, he showed mild aortic valve stenosis and insufficiency. Implantation time (time between implantation and explantation) of CardioCel patches was a median of 25 (range 11-47) months. Explants were examined using a uniform protocol with methylmethacrylate and/or paraffin embedding after fixation in formalin. Besides standard histological staining, von Kossa (for identification of calcifications) and immunohistochemical stains were applied with antibodies specific for muscular, inflammatory and connective tissue component antigens. Findings regarding the extent of appositional growth on top of the patch consisting of fibroblasts and extracellular matrix components, calcification, and inflammation were rated using a 4-grade scale (G0 no/G1 few/G2 moderate/G3 massive). RESULTS: Superficial endothelialization was demonstrated in all patients by immunohistochemical analysis. Nine specimens showed mild inflammatory cell infiltration (G1) (G2: n = 3). Significant appositional growth on top of the patch due to addition of fibroblasts and extracellular matrix components, was seen in all specimens (G1: n = 1; G2: n = 7; G3: n = 4). Ten of 12 samples with implant times of 23 months or longer revealed calcifications (G1: n = 6; G2: n = 3; G3: n = 1). Two specimens with the shortest implant times (11 and 20 months) showed no calcifications (G0). Thrombus apposition with granulocyte infiltration was demonstrated in the specimen of the patient with endocarditis. CONCLUSIONS: In our cohort, all CardioCel patches used for aortic valve repair in patients with congenital heart disease demonstrated appositional growth of fibroblasts and extracellular matrix components, and calcification after an implant time of at least 23 months.

Subclinical thrombus formation in bioprosthetic pulmonary valve conduits.

OBJECTIVES: Bioprosthetic pulmonary valve conduits have been reported with an increased risk of endocarditis. Thrombus formation is considered as source of these serious and life-threatening infections. We reviewed a series of explanted valved pulmonary conduits for histological evidence for thrombus formation. MATERIALS AND METHODS: Explanted bioprosthetic pulmonary valves were fixed in formalin and embedded in paraffin or in methylmethacrylate. Standard staining as well as immunohistochemical staining techniques were applied. Native pulmonary valves of German domestic pigs served as controls. RESULTS: 47 valved pulmonary conduits (Hancock n = 23, Homograft n = 7, Contegra n = 7, Melody n = 7, other n = 3) were analyzed histologically. Average time of implantation had been 63 months (6 to 342 months). Indications for explantation included significant obstruction (n = 45), regurgitation (n = 7), and/or endocarditis (n = 6). In 44/47 (93%) specimen, we found accumulation of thrombotic material at the basis of the semilunar valve sinus to a variable degree. 11 patients had been treated with antiplatelet agents, 2 had received anticoagulants at the time of explantation. There was no suspicion of thrombus formation clinically or echocardiographically prior to explantation in any of the patients. Control porcine pulmonary valves (n = 5) did not show any evidence of accumulation of thrombotic material. CONCLUSIONS: In a large series of explanted valved pulmonary conduits, formation of subclinical, mostly non-infectious thrombotic material was an almost ubiquitous finding. We speculate that high incidence of endocarditis in bioprosthetic valves may in part be explained by thrombus apposition at the basis of conduit valve sinus.

Thrombosis of a mechanical prosthetic aortic valve in early pregnancy: histopathological findings.

We present results of the histopathological workup of a prosthetic aortic valve which was explanted from a 31-year-old woman due to valve thrombosis during early pregnancy. Our images demonstrate nicely thrombotic material directly adherent to the pyrolytic carbon surface in a human specimen.

Melody transcatheter valve: Histopathology and clinical implications of nine explanted devices.

OBJECTIVES: We examined interventionally implanted valved Melody conduits after surgical explantation by means of histology and immunohistochemistry and matched these findings with clinical data in order to assess in vivo biocompatibility and to identify risk factors for graft failure. METHODS: 9 Melody valves had been implanted in 8 patients (pulmonary n = 7, tricuspid position n = 1). Indication for explantation included significant obstruction in 7 patients and valve insufficiency in 1 patient. 4 of 8 patients had suffered from endocarditis. Mean interval between implantation and explantation was 3.2 (1.8-5.2) years. All explants were worked up using a uniform protocol with fixation in formalin and embedding in methylmethacrylate. RESULTS: All but one valve of the explanted Melody grafts were thin and histologically intact without any pathological findings. Complete neo-endothelialization could be demonstrated by means of immunohistochemistry. All 4 Melody valves from patients with endocarditis showed dense granulocytic infiltrations, 3 of these showed thrombotic material within the valves. CONCLUSION: This report covers the first series of explanted Melody valves from humans applying a uniform protocol for histopathological examination. Good biocompatibility of the Melody valves could be demonstrated after a mid-term follow-up. Factors for graft failure included endocarditis, outgrowth, and residual stenosis. These findings may have significant implications for the implant procedure as well as care of the patients during long-term follow-up.

A Comparative Histopathological Study of Heparin Coated and Uncoated Polytetrafluoroethylene Shunts in Children With Congenital Heart Defect.

OBJECTIVE: Recently, heparin coated polytetrafluoroethylene (PTFE) shunts are available and are believed to improve inherent shunt problems such as thrombosis and excessive and incomplete neointima formation or occlusion. We aimed at comparing the potential histopathological differences in the neointima (in) between uncoated (UCS) PTFE shunts and heparin coated (HCS) PTFE shunts. MATERIALS AND METHODS: Thirteen shunts (six UCS and seven HCS) were analyzed. The specimens were fixed in formalin, embedded in paraffin or in methylmethacrylate, and characterized by standard and immunohistochemical staining. The thickness of pseudointima proliferation was graded as follows: 0 = no cell layers, 1 = few layers <100 µm, 2 = partial layers >100 µm, 3 = complete layers <300 µm, 4 = complete layers >300 µm, and 5 = occlusion. RESULTS: Mean shunt size was 3.4 ± 0.2 mm in UCS and 3.1 ± 0.2 mm in HCS (P = .053). Mean time of implantation was 163 ± 75 days in UCS and 97 ± 52 days in HCS (P = .091). There were no significant differences in the proportion of patients with functionally single ventricle, body surface area, age at implantation, or implantation type, between both groups. Shunt occlusion did not occur. Unplanned shunt explantation due to cyanosis was performed in one patient in each group. Partial thrombus formation was observed in one UCS (P = .462). There was complete endothelialization in 50% of UCS and 86% of HCS (P = .266). The grade of pseudointima proliferation was 1.8 ± 0.4 in UCS and 1.7 ± 0.5 in HCS (P = .646). CONCLUSIONS: The histopathological workup of PTFE shunts revealed equally partial endothelialization and discrete pseudointima proliferation in both the groups. The process of endothelialization may be faster in HCS.

Clinical, echocardiographic and histopathologic findings in nine patients with surgically explanted ASD/PFO devices: do we know enough about the healing process in humans?

BACKGROUND: Atrial septal defects (ASD) and persistent foramen ovale (PFO) are managed in increasing numbers by catheter interventions as an attractive alternative to surgery. Early complications have been described in clinical series whereas late complications are rare. No series are reported with clinical, echocardiographic and histological data. METHODS AND RESULTS: We collected clinical, echocardiographic, and histolological data of nine patients with surgically explanted devices. Occlusion devices were explanted after a mean interval of 3.4 ± 2.4 years (range 0.9-8.3). Indications were recurrent thromboembolic events in five, residual shunt/dislocation in three, and growing mass on echocardiography despite oral anticoagulation in one patient. Two patients suffered potentially live threatening events due to coronary embolism. One of them had to be resuscitated due to ventricular fibrillation. Histologically, residues of superficial thrombus formation could be demonstrated in two of the devices. In another patient, hyperplastic tissue formation was related to a local inflammatory process but not to a thrombus as suspected by echocardiography. CONCLUSION: Late complications after device implantation may occur up to 8 years after device implantation and may be potentially live threatening. Echocardiographic controls should be prolonged beyond the first year after implantation and every explanted device should be histologically worked up in an experienced center. Up to now, the mechanisms of late thrombogenesis are not fully understood.

Immunohistochemical characterization of neotissues and tissue reactions to septal defect-occlusion devices.

BACKGROUND: We sought to evaluate tissue reactions within and at the surface of devices for interventional therapy of septal defects and to identify antigen characteristics of neotissues. METHODS AND RESULTS: Atrial or ventricular septal defect-occlusion devices (Amplatzer, n=7; Cardioseal/Starflex, n=3) were processed using a uniform protocol after surgical removal from humans (implantation time, 5 days to 4 years). Devices were fixed in formalin and embedded in methylmethacrylate. Serial sections were obtained by sectioning with a diamond cutter and grinding, thus saving the metal/tissue interface for histologic evaluation. Immunohistochemical staining was performed using conventional protocols. Superficial endothelial cells stained positive for von Willebrand factor. Within the newly formed tissues, fibroblast-like cells were identified with a time-dependent expression of smooth muscle cell maturation markers (smooth muscle actin, smooth muscle myosin, h-caldesmon, and desmin) beside extracellular matrix components. Neovascularization of the newly formed tissues was demonstrated with the typical immunohistochemical pattern of capillaries and small vessels. Inflammatory cells could be identified as macrophages (CD68+) and both T-type and B-type lymphocytes (CD3+, CD79+). CONCLUSIONS: This is the first presentation of results from serial immunohistochemical staining of a collection of explanted human septal-occlusion devices. A time-dependent maturation pattern of the fibroblast-like cells in the neotissues around the implants could be described. Neoendothelialization was seen in all specimens with implantation times of 10 weeks or more. The time course of neoendothelialization, as seen in our study, further supports the clinical practice of anticoagulant or antiplatelet therapy for 6 months after implantation. This time interval should be sufficient to prevent thromboembolic events due to thrombus formation at the foreign surface of cardiovascular implants.

A novel method for processing resin-embedded specimens with metal implants for immunohistochemical labelling.

A major technical problem in the processing of resin-embedded tissues is the adhesion of the tissue sample on glass slides for immunohistochemical labelling. We therefore established a novel protocol for processing such specimens with improved attachment of the tissue sample during resin removal (deplastification). In order to demonstrate the feasibility of the procedure we employed a panel of smooth muscle cell maturation markers. The technique makes use of a silicone glue (Elastosil E41; Wacker Chemie, München, Germany) to attach the tissue samples to the glass slides. This allows resin dissolution in xylene/2-methoxyethylacetate without detachment of the sample from the slide. Our results demonstrate successful immunohistochemical labelling with primary antibodies directed against: smooth muscle actin, smooth muscle myosin, h-caldesmon, desmin, vimentin and von Willebrand factor. In conclusion, we have established a new and successful method for resin-embedded sample adhesion on glass slides. The developed protocol is feasible for investigation of cells which are involved in intimal proliferation following stent implantation.