Endocannabinoid analysis in GlucoEXACT plasma: Method validation and sample handling recommendations.

Endocannabinoids (ECs), such as anandamide and 2-arachidonyl glycerol (2-AG), contribute to the pathology of inflammatory, malignant, cardiovascular, metabolic and mental diseases. The reliability of quantitative analyses in biological fluids of ECs and endocannabinoid-like (EC-like) substances depends on pre-analytical conditions such as temperature and "time-to-centrifugation". Standardization of these parameters is critical for valid quantification and implementation in clinical research. In this study, we compared concentrations obtained with GlucoEXACT blood collection tubes versus K3EDTA tubes and employed the optimized procedure to assess ECs profiles in patients with inflammatory skin disease and healthy controls. A UHPLC-MS/MS method was validated for human plasma from GlucoEXACT blood collection tubes according to EMA and FDA guidelines, and pre-analytical conditions were systematically modified to assess analyte stability and optimize the procedures. The results showed significantly lower concentrations of ECs and EC-like substance concentrations with GlucoEXACT tubes compared with K3EDTA tubes, and GlucoEXACT extended the time window of stable concentrations. The strongest method-disagreement occurred for 1/2-AG suggesting that GlucoEXACT delayed ex vivo isomer rearrangement. Hence, GlucoExact tubes were superior in terms of stability and reliability. However, although absolute concentrations obtained with GlucoExact and K3EDTA differed, linear regression studies showed high agreement (except for 1/2-AG), and both methods showed similar EC profiles and similar disease-dependent pro-inflammatory patterns in dermatology patients. Hence, despite the obstacles in EC analyses, implementation of optimized pre-analytical blood collection and sample processing procedures provide reliable insight into peripheral ECs.

Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.

The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

Post-mortem changes of prostanoid concentrations in tissues of mice: Impact of fast cervical dislocation and dissection delay.

Prostanoids are potent lipid mediators involved in a wide variety of physiological functions like blood pressure regulation or inflammation as well as cardiovascular and malign diseases. Elucidation of their modes of action is mainly carried out in pre-clinical animal models by quantifying prostanoids in tissues of interest. Unfortunately, prostanoids are prone to post-mortem artifact formation and de novo synthesis can already be caused by external stimuli during the euthanasia of animals like prolonged hypercapnia or ischemia. Therefore, this study investigates the suitability and impact of fast cervical dislocation for the determination of prostanoids (6-keto-PGF1α, TXB2, PGF2α, PGD2, PGE2) in seven tissues of mice (spinal cord, brain, sciatic nerve, kidney, liver, lung, and spleen) to minimize time-dependent effects and approximate physiological concentrations. Tissues were dissected in a standardized sequence directly or after 10 min to investigate the influence of dissection delays. The enzyme inhibitor indomethacin (10 µM) in combination with low processing temperatures was employed to preserve prostanoid concentrations during sample preparation. Quantification of prostanoids was performed via LC-MS/MS. This study shows, that prostanoids are differentially susceptible to post-mortem artifact formation which is closely connected to their physiological function and metabolic stability in the respective tissues. Prostanoids in the brain, spinal cord, and kidney that are not involved in the regulatory response post-mortem, i.e. blood flow regulation (6-keto-PGF1α, PGE2, PGF2α) showed high reproducibility even after dissection delay and could be assessed after fast cervical dislocation if prerequisites like standardized pre-analytical workflows with immediate dissection and inhibition of residual enzymatic activity are in place. However, in tissues with high metabolic activity (liver, lung) more stable prostanoid metabolites should be used. Moreover, prostanoids in the spleen were strongly affected by dissection delays and presumably the method of euthanasia itself.

Pre-analytical challenges for the quantification of endocannabinoids in human serum.

Endocannabinoids (ECs) are potent lipid mediators with high physiological relevance. They are involved in a wide variety of diseases like depression or multiple sclerosis and are closely connected to metabolic parameters in humans. Therefore, their suitability as a biomarker in different (patho-)physiological conditions is discussed intensively and predominantly investigated by analyzing systemic concentrations in easily accessible matrices like blood. Carefully designed pre-analytical sample handling is of major importance for high-quality data, but harmonization is not achieved yet. Whole blood is either processed to serum or plasma before the onset of analytical workflows and while knowledge about pre-analytical challenges in plasma handling is thorough they were not systematically investigated for serum. Therefore, the ECs AEA and 2-AG, and closely related EC-like substances 1-AG, DHEA, and PEA were examined by LC-MS/MS in serum samples of nine healthy volunteers employing different pre-analytical sample handling protocols, including prolonged coagulation, and storage after centrifugation at room temperature (RT) or on ice. Furthermore, all analytes were also assessed in plasma samples obtained from the same individuals at the same time points to investigate the comparability between those two blood-based matrices regarding obtained concentrations and their 2-AG/1-AG ratio. This study shows that ECs and EC-like substances in serum samples were significantly higher than in plasma and are especially prone to ex vivo changes during initial and prolonged storage for coagulation at RT. Storage on ice after centrifugation is less critical. However, storage at RT further increases 1-AG and 2-AG concentrations, while also lowering the already reduced 2-AG/1-AG ratio due to isomerization. Thus, avoidance of prolonged processing at RT can increase data quality if serum as the matrix of choice is unavoidable. However, serum preparation in itself is expected to initiate changes of physiological concentrations as standard precautionary measures like fast and cooled processing can only be utilized by using plasma, which should be the preferred matrix for analyses of ECs and EC-like substances.

Presence of Escherichia coli, Trueperella pyogenes, α-hemolytic streptococci, and coagulase-negative staphylococci and prevalence of subclinical endometritis.

Subclinical endometritis (SCE) has a severe negative effect on reproductive performance of dairy cows. Different studies have shown a wide variety of cut points for the polymorphonuclear neutrophilic leukocyte (PMNL) percentage to define SCE and differing sampling times. Possible risk factors for SCE, such as low body condition score, hyperketonemia, and increased haptoglobin concentrations have been demonstrated. The aim of this study was to determine the relationship between bacterial findings in the uterus and PMNL in the early postpartum period and to compare 2 classification schemes for the diagnosis of SCE. Cytological and bacteriological samples from the uterus were taken from 149 cows at 2 consecutive times postpartum [10 ± 1 and 24 ± 1 d in milk (DIM)] and additionally, 131 cows were sampled at 21 to 27 DIM. For sample collection, the cytobrush technique was used and vaginal discharge was examined by vaginoscopy. Polymorphonuclear neutrophilic leukocyte cut points were set at 5, 10, and 18%. Cows positive with α-hemolytic streptococci at 10 ± 1 DIM had significant higher PMNL percentages at 21 to 27 DIM and greater median days to pregnancy (193.5d) than cows not infected (123.0 d). Cows with Trueperella pyogenes at 10±1 DIM had higher odds (5.2) of having more than 18% PMNL at 21 to 27 DIM compared with cows without infection. Escherichia coli had no effect on the prevalence of SCE regardless of the cut point. Primiparous cows with more than 18% PMNL had greater median days to pregnancy (144.5 d) than cows with less than 5% (80.0 d) or cows between 5 and 18% PMNL (68.0 d). Cows between 5 and 18% PMNL or 10 and 18% PMNL showed the best reproductive performance. The results demonstrate that an early infection with α-hemolytic streptococci increased the PMNL percentage 2 wk later and that 18% PMNL at 21 to 27 DIM was a predictive cut point for primiparous cows to diagnose animals with or without SCE.

[Acute and fulminant fungal sinusitis in immunosuppressed patients]. / Die akute invasive und fulminant verlaufende Pilzsinusitis bei immunsupprimierten Patienten.

BACKGROUND: Acute and invasive fungal sinusitis represent rare diseases which can lead to life threatening complications. Immunosuppressed patients are affected primarily. The expansion of transplantation medicine and the progress in therapy of malignant diseases of the lymphatic system are associated with an increase of opportunistic systemic mycoses. Therefore the otorhinolaryngologist is confronted increasingly with these problems and questions for surgery, especially if the symptom of a periorbital inflammation occurs as a sign for a beginning orbital complication and radiological signs of involvement of the paranasal sinuses exist. PATIENTS: We report exemplary about two immunosuppressed patients with an invasive and fulminant fungal aspergillosis of the paranasal sinuses. In spite of systemic antifungal therapy and surgical intervention, intracranial and systemic complications caused a lethal course. CONCLUSIONS: In immunosuppressed patients with clinical and radiological signs for a sinusitis and a periorbital inflammation an invasive fungal sinusitis should be considered. Pathogenic aspergillus species are the most common identified in fungal sinusitis. The disease with often lethal outcome requires a careful and fast diagnostic and therapy as well as interdisciplinary co-operation. If and when limited or extensive surgery should be performed remains, because of the rarity and the lacking experience with this disease, still a controversially discussed issue and depends on several factors: the kind of disease, the immunity, the subtype of invasive fungal sinusitis and the degree of tissue invasion.

[Degenerative changes in parapharyngeal schwannoma "ancient schwannoma". Case report and review of the literature]. / Degenerativ verändertes parapharyngeales Schwannom ,,ancient schwannoma". Fallbericht und Literaturübersicht.

Schwannomas are rare, slow-growing, benign neoplasms derived from the schwann cells of the nerve sheath of peripheral nerves. Extracranial schwannomas are most frequently localized within the extremities or the head and neck region. For example, they are common in the skull base, the carotid sheath, and the parapharyngeal space. Beneath several histological variants, schwannomas showing prominent degenerative changes (so-called,,ancient schwannomas") are exceedingly rare. Histopathological features, such as degenerative changes and nuclear atypias in ancient schwannomas, may be easily confused with malignant mesenchymal neoplasm.We present the case of a 32-year-old female who required surgery for a parapharyngeal neoplasm. Histological examination revealed features of an ancient schwannoma.

Frequency dependence of sonophoresis.

PURPOSE: Application of low-frequency ultrasound has been shown to increase skin permeability, thereby facilitating delivery of macromolecules (low-frequency sonophoresis). In this study, we sought to determine the dependence of low-frequency sonophoresis on ultrasound frequency, intensity and energy density. METHODS: Pig skin was exposed to low-frequency ultrasound over a range of ultrasound frequency and intensity conditions. The degree of skin permeabilization was measured using its conductivity. Imaging experiments were also carried out to visualize the transport pathways created by ultrasound. RESULTS: The data showed that for each frequency (in the range of 19.6-93.4 kHz), there exists a threshold intensity below which no detectable conductivity enhancement was observed. The threshold intensity increased with frequency. It is feasible to achieve the desired conductivity (permeability) enhancement regardless of the choice of frequency, although the necessary energy density is higher at higher frequencies. Low frequencies (approximately 20 kHz) induced localized transport compared to a more dispersed effect seen with higher frequencies (approximately 58.9 kHz). CONCLUSIONS: This study provides a quantitative understanding of the effects of low-frequency ultrasound on skin permeability.

Demonstration of cytokeratins by immunoperoxidase staining in prostatic tissue.

The presence and distribution of cytokeratins (CK) have been investigated using an epidermal keratin antiserum in various dilutions and the PaP (peroxidase-antiperoxidase) and avidin-biotin-peroxidase (ABC) immunohistochemical methods. A total of 44 samples of prostatic tissue were divided into alcohol-fixed (22 cases) and formaldehyde-fixed (22 cases). Each group included 12 non-malignant lesions (hyperplasias and prostatitis) and 10 adenocarcinomas. The best results were achieved with the ABC method in alcohol-fixed tissues, while formaldehyde-fixed tissues gave poor staining despite the use of different enzymes to unmask antigenic determinants. With similar dilutions of the specific antiserum the PaP method gave less intense staining. Cytokeratins were detected in basal and columnar cells, in areas of transitional and squamous metaplasia and in normal transitional epithelium. Columnar cells showed strong staining in the supranuclear portion. Adenocarcinomas gave positive staining for cytokeratins varying from weak to strong. The intensity of staining showed no correlation with the degree of differentiation of the tumor. Different degrees of intensity were frequently observed within the same tumor. High dilutions of the specific antiserum (greater than 1/400) failed to stain carcinomas or stained them poorly, whereas they still stained normal or hyperplastic tissues. Gland-forming tumors showed a highly polarized labelling with the strongest staining in the luminal portion of the cell. The conclusion is that all epithelial prostatic tissues, benign and malignant, express cytokeratins.

Full duplex transmission link over single-strand optical fiber.

Full duplex transmission of television signals over a single optical fiber is demonstrated. The transmission link uses directional couplers that possess high directivity and low insertion loss.

Optical interpolation with application to array processing.

The use of spatial filtering for optical interpolation between sample values of a function is studied. Onedimensional interpolation between channels of density or area modulated spatial signals is analyzed and the effect of nonzero sample width presented. A new bound is given for the error due to the number of samples being finite. It is shown how exact optical interpolation of periodic functions may be obtained from the samples of only one period by the use of a diffraction grating in the frequency plane. An experiment demonstrates the application of interpolation to processing signals from a simulated sensor array.