A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules

Pathological Findings in Male and Female Semi-Professional Football Players from 11 to 14 Years—A Report of the Bavarian Football Association’s Pre-Participation Screening Program

Pre-participation screening (PPS) in professional junior football is common practice. However, football players (FP) from non-professional football clubs may also be exposed to health risks, both internal and musculoskeletal. Therefore, the Bavarian Football Association (BFV) implemented a cardiological and orthopedic screening program for semi-professional FP in 2014. The purpose of this study was to obtain and present epidemiological data of pre-adolescent and adolescent semi-professional FP, including cardiac pathologies, past injuries, and orthopedic disorders. This study represents a retrospective analysis of semi-professional FP aged 11 to 14 years participating in the PPS program from 2014 to 2018, including their medical history, cardiac risk profiles, and the results of undergoing orthopedic and sports cardiology examinations. Overall, 362 male and 162 female FP could be included. More than 20% of the FP indicated suffering from one or more medical conditions. Cardiac abnormalities were reported in 30 (5.7%) FP. Further cardiological diagnostics were recommended for 3% of the FP due to findings while undergoing the PPS. Orthopedic disorders could be detected in 51 (9.7%) FP. Of the reported injuries, 44.3% could be categorized as overuse injuries. In order to guarantee extensive preventive sports medical care for semi-professional junior FP, a PPS concept should include a basic orthopedic examination in addition to cardiological screening due to a high rate of overuse injuries and cardiac abnormalities among pre-adolescent and adolescent FP. Further studies are needed in junior football to gain epidemiological data on injury occurrence and cardiac abnormalities on an amateur level to evaluate possible PPS programs, even on an amateur level

Pathological Findings in Male and Female Semi-Professional Football Players from 11 to 14 Years—A Report of the Bavarian Football Association’s Pre-Participation Screening Program

Pre-participation screening (PPS) in professional junior football is common practice. However, football players (FP) from non-professional football clubs may also be exposed to health risks, both internal and musculoskeletal. Therefore, the Bavarian Football Association (BFV) implemented a cardiological and orthopedic screening program for semi-professional FP in 2014. The purpose of this study was to obtain and present epidemiological data of pre-adolescent and adolescent semi-professional FP, including cardiac pathologies, past injuries, and orthopedic disorders. This study represents a retrospective analysis of semi-professional FP aged 11 to 14 years participating in the PPS program from 2014 to 2018, including their medical history, cardiac risk profiles, and the results of undergoing orthopedic and sports cardiology examinations. Overall, 362 male and 162 female FP could be included. More than 20% of the FP indicated suffering from one or more medical conditions. Cardiac abnormalities were reported in 30 (5.7%) FP. Further cardiological diagnostics were recommended for 3% of the FP due to findings while undergoing the PPS. Orthopedic disorders could be detected in 51 (9.7%) FP. Of the reported injuries, 44.3% could be categorized as overuse injuries. In order to guarantee extensive preventive sports medical care for semi-professional junior FP, a PPS concept should include a basic orthopedic examination in addition to cardiological screening due to a high rate of overuse injuries and cardiac abnormalities among pre-adolescent and adolescent FP. Further studies are needed in junior football to gain epidemiological data on injury occurrence and cardiac abnormalities on an amateur level to evaluate possible PPS programs, even on an amateur level

Echocardiographic diagnosis of congenital coronary artery abnormalities in a continuous series of adolescent football players

Background Sudden cardiac death (SCD) in children and adolescents is rare. Several studies have reported a higher risk of SCD during athletic competition. High risk congenital coronary artery abnormalities are the second leading cause of SCD in young athletes in the USA. Echocardiographic assessment of coronary arteries has not been routinely used in screening programmes for junior athletes so far. Design Prospective cohort study in 1045 consecutive adolescent elite football players. Methods All athletes underwent a standardized cardiovascular screening protocol with a medical history, a physical examination, 12-lead resting electrocardiogram and a complete transthoracic 2D-echocardiography. Results Two athletes (0.19%) showed a high-risk coronary artery abnormality (CAA) with a right coronary artery originating abnormal from the aorta and coursing inter-arterial. Low-risk CAAs were found in 16 athletes (1.53%). There was an ectasia of the left coronary artery (+3.9z and +4.3z) and a fistula from the left coronary artery in two cases (0.19%), respectively. In 1.05% (n = 11) we found a high take-off (2.3-6.8 mm) and in one case (0.096%) there was a tangential take-off of the right main coronary artery. Variants of coronary arterial anatomy were identified in 335 of 1045 athletes (32.06%). Conclusion Basic pre-participation screening tests including 12-lead or exercise electrocardiogram do not safely identify high-risk CAAs. In adolescent athletes an expert cardiologist is able to describe the origin and the proximal course of the coronary arteries and identify major abnormalities in most of the cases by transthoracic 2D-echocardiography

Pathological Findings in Male and Female Semi-Professional Football Players from 11 to 14 Years—A Report of the Bavarian Football Association’s Pre-Participation Screening Program

Pre-participation screening (PPS) in professional junior football is common practice. However, football players (FP) from non-professional football clubs may also be exposed to health risks, both internal and musculoskeletal. Therefore, the Bavarian Football Association (BFV) implemented a cardiological and orthopedic screening program for semi-professional FP in 2014. The purpose of this study was to obtain and present epidemiological data of pre-adolescent and adolescent semi-professional FP, including cardiac pathologies, past injuries, and orthopedic disorders. This study represents a retrospective analysis of semi-professional FP aged 11 to 14 years participating in the PPS program from 2014 to 2018, including their medical history, cardiac risk profiles, and the results of undergoing orthopedic and sports cardiology examinations. Overall, 362 male and 162 female FP could be included. More than 20% of the FP indicated suffering from one or more medical conditions. Cardiac abnormalities were reported in 30 (5.7%) FP. Further cardiological diagnostics were recommended for 3% of the FP due to findings while undergoing the PPS. Orthopedic disorders could be detected in 51 (9.7%) FP. Of the reported injuries, 44.3% could be categorized as overuse injuries. In order to guarantee extensive preventive sports medical care for semi-professional junior FP, a PPS concept should include a basic orthopedic examination in addition to cardiological screening due to a high rate of overuse injuries and cardiac abnormalities among pre-adolescent and adolescent FP. Further studies are needed in junior football to gain epidemiological data on injury occurrence and cardiac abnormalities on an amateur level to evaluate possible PPS programs, even on an amateur level

The PAXgene^® Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research

<div><p>Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.</p> </div

Proteins extracted from PFPE tissues are suitable for ELISAs with antibodies directed against denatured proteins.

<p>Three human malignant (breast cancer metastasis, ovarian and prostate cancer) and three non-malignant (stomach, duodenum, uterus) tissue specimens were each divided into two samples and either cryopreserved (cryo) or fixed and stabilized in the PAXgene Tissue reagents and paraffin-embedded (PFPE). Proteins were extracted with respective protocols (See ELISA, Experimental Section) using denaturing or non-denaturing extraction buffer. Results are always depicted in duplicates for each lysate. (<b>A</b>) An ELISA for non-denatured Akt (n = 2 for stomach and duodenum samples. (<b>C</b>) An ELISA for denatured Erk-1/2 (n = 2). (<b>E</b>) An ELISA for denatured phospho-Erk-1/2 (n = 3). Western blot analysis of (<b>B)</b> 12.5 µg or (<b>D, F</b>) 25 µg protein of each lysate separated by SDS-PAGE and performed using indicated antibodies.</p

Proteins extracted from PFPE tissues are suitable for two-dimensional gel electrophoresis.

<p>Two human non-malignant (duodenum, ovary) tissue specimens were each divided into three samples and either cryopreserved (cryo), fixed and stabilized in the PAXgene Tissue reagents and paraffin-embedded (PFPE) or fixed in formalin and paraffin-embedded (FFPE). Proteins were extracted with respective protocols (See Two-dimensional SDS-PAGE, Experimental Section) and 150 µg protein of each was separated by two-dimensional SDS-PAGE. The isoelectric focusing was conducted in a range between pH 3–pH 10.</p