Investigations into the concentrations and metabolite profiles of doping agents and antidepressants in human seminal fluid using liquid chromatography-mass spectrometry.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called Adverse Analytical Finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offences and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from non-athletes were analysed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e. samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the non-approved PPARδ agonist GW1516) in a total of 81 samples, with 91 % of these suspected cases being verified by the confirmation method. Besides the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. Significance Statement This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

Evaluation of university and training standards in clinical perfusion, an European-wide survey.

OBJECTIVES: Adequate theoretical and practical training of prospective clinical perfusionists is essential for maintaining clinical standards and ensuring patient safety during cardiac surgery procedures. Perfusion schools play a crucial role in establishing and maintaining higher education and training standards in clinical perfusion. The aim of this study is to obtain a comprehensive overview of European training standards in clinical perfusion in 2023. METHODS: For this study, 53 perfusion schools in Europe were found and contacted, of which 30 (56.6%) responded, giving a sample size of n = 30, which were then included in the data analysis. The quantitative data of the survey are analysed using descriptive methods. RESULTS: The university and training standards in clinical perfusion in Europe vary in many respects. Starting with the entry criterion for studies (most frequently a required bachelor's degree 36.7% or 2nd most common an university entrance qualification 30%), the duration [from <12 months (13.3%) up to 36 months (13.3%)] and regarding the content of the teaching in clinical perfusion [<30 European Credit Transfer System (ECTS) (33.3%) and more than 180 ECTS (6.7%)]. The mean value for teaching in clinical perfusion content is 62.63 ECTS credits. CONCLUSIONS: The obtained results show important differences between countries and schools. As such, they form a valuable database for future discussions establishing a common European curriculum and training standards for perfusionists. For the generalizability of the results, further evaluations and larger samples are needed.

Longitudinal evaluation of laboratory results and method precision in worldwide erythropoietin external quality assessments.

Introduction: This study presents a longitudinal analysis of external quality assessment (EQA) results for erythropoietin (EPO) determinations conducted between 2017 and 2022 with a continuously increasing number of participating laboratories. The aim of this work was to evaluate participant performance and methodological aspects. Methods: In each of the eleven EQA surveys, a blinded sample set of lyophilized human serum containing one sample with lower EPO concentrations (L) and one with higher EPO concentrations (H) was sent to the participating laboratories. Results: A total of 1,256 measurements were included. The median (interquartile range) fraction of participants not meeting the criteria of acceptance set at 20% around the robust mean of the respective survey was 9.5% (6.1%-10.7%) (sample L) and 9.1% (5.8%-11.8%) (sample H) but lacked a clear trend in the observed period. Some surveys exhibited unusually high interlaboratory variation, suggesting interfering components in the EQA samples. Different immunological methods and reagent manufacturers also showed variability in measurement outcomes to some extent. Conclusion: These findings highlight the need for continuous quality assessment in EPO measurements to ensure patient safety and identify areas for further research and investigation.

Central and Peripheral Circulation Differ during Off-Pump Coronary Artery Bypass Grafting.

Background: Off-pump coronary artery bypass grafting (OPCAB) is an alternative to on-pump coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB). During OPCAB, the temporary use of an intracoronary shunt and inotropic medication or catecholamines should keep the central hemodynamics constant. Nevertheless, the need for conversion to on-pump CABG often occurs unexpectedly, most likely due to circulation instability. Circulation instability can appear first in peripheral body parts; therefore, peripheral microcirculation might serve as a predictor for the upcoming conversion to on-pump CABG. We investigated the impact of coronary artery ligation and shunt insertion during OPCAB on cutaneous microcirculation (cLDP) with Laser Doppler Perfusion Technology and transcutaneous oxygen partial pressure ( tcpO 2 ). Methods: In a pig model of OPCAB, peripheral circulation was evaluated after cLDP (N = 17) and tcpO 2 (N = 6) monitoring. Systolic, diastolic, and mean arterial pressure were also observed to prove the independence of perfusion measurement results from hemodynamic parameters. Results: Ligation time during cLDP and tcpO 2 monitoring were 101 ± 49 s and 83 ± 33 s, respectively. Shunt time was 11 ± 3 min during cLDP and 13 ± 2 min during tcpO 2 measurement. Ligation of the left anterior descending coronary artery (LAD) reduced cLDP significantly to 88 ± 14% (p = 0.007) and tcpO 2 to 71 ± 25% (p = 0.038). Inserting a temporary shunt into the LAD significantly improved cLDP (p = 0.006) and tcpO 2 (p = 0.015) compared to ligation. cLDP was restored to 99%, and tcpO 2 was restored to 91% of the baseline level before ligation. All hemodynamic parameters remained stable and did not change significantly during OPCAB. Conclusions: Although hemodynamic parameters stayed constant, peripheral microcirculation was influenced markedly during OPCAB. Inserting a temporary shut into the LAD leads to a complete normalization of peripheral microcirculation, regarding evaluation by cLDP and tcpO 2 .

Longitudinal analysis of external quality assessment of immunoassay-based steroid hormone measurement indicates potential for improvement in standardization.

As hormonal disorders are linked to several diseases, the accurate quantitation of steroid hormone levels in serum is crucial in order to provide patients with a reliable diagnosis. Mass spectrometry-based methods are regarded as having the highest level of specificity and sensitivity. However, immunoassays are more commonly used in routine diagnostics to measure steroid levels as they are more cost effective and straightforward to conduct. This study analyzes the external quality assessment results for the measurement of testosterone, progesterone and 17ß-estradiol in serum using immunoassays between early 2020 and May 2022. As reference measurement procedures are available for the three steroid hormones, the manufacturer-specific biases were normalized to the reference measurement values. The manufacturer-specific coefficients of variation were predominantly inconspicuous, below 20% for the three hormones when outliers are disregarded, however there were large differences between the various manufacturer collectives. For some collectives, the median bias to the respective reference measurement value was repeatedly greater than ±35%, which is the acceptance limit defined by the German Medical Association. In the case of testosterone and progesterone determination, some collectives tended to consistently over- or underestimate analyte concentrations compared to the reference measurement value, however, for 17ß-estradiol determination, both positive and negative biases were observed. This insufficient level of accuracy suggests that cross-reactivity continues to be a fundamental challenge when antibody detection is used to quantify steroids with a high structural similarity. Distinct improvements in standardization are required to provide accurate analysis and thus, reliable clinical interpretations. The increased accuracy of the AX immunoassay for testosterone measurement, as observed in the INSTAND EQAs between 2020 and 2022, could be the result of a recalibration of the assay and raises hope for further improvement of standardization of immunoassay-based steroid hormone analyses in the coming years.

Improving Diastolic and Microvascular Function in Heart Transplantation with Donation after Circulatory Death.

The impact of the machine perfusion of donation after circulatory death (DCD) hearts with the novel Custodiol-N solution on diastolic and coronary microvascular dysfunction is unknown. Porcine DCD-hearts were maintained four hours by perfusion with normothermic blood (DCD-B), hypothermic Custodiol (DCD-C), or Custodiol-N (DCD-CN), followed by one hour of reperfusion with fresh blood, including microvascular and contractile evaluation. In another group (DCD group), one hour of reperfusion, including microvascular and contractile evaluation, was performed without a previous maintenance period (all groups N = 5). We measured diastolic function with a balloon catheter and microvascular perfusion by Laser-Doppler-Technology, resulting in Laser-Doppler-Perfusion (LDP). We performed immunohistochemical staining and gene expression analysis. The developed pressure was improved in DCD-C and DCD-CN. The diastolic pressure decrement (DCD-C: -1093 ± 97 mmHg/s; DCD-CN: -1703 ± 329 mmHg/s; DCD-B: -690 ± 97 mmHg/s; p < 0.05) and relative LDP (DCD-CN: 1.42 ± 0.12; DCD-C: 1.11 ± 0.13; DCD-B: 1.22 ± 0.27) were improved only in DCD-CN. In DCD-CN, the expression of eNOS increased, and ICAM and VCAM decreased. Only in DCD-B compared to DCD, the pathways involved in complement and coagulation cascades, focal adhesion, fluid shear stress, and the IL-6 and IL-17 pathways were upregulated. In conclusion, machine perfusion with Custodiol-N improves diastolic and microvascular function and preserves the microvascular endothelium of porcine DCD-hearts.

Role of Lp(a) in patients with erectile dysfunction undergoing angioplasty for symptomatic pelvic artery disease.

Background: Atherosclerotic disease of erection-related arteries is a major reason for erectile dysfunction (ED). Lp(a) has been implied in the pathophysiology of atherosclerosis in the coronary and lower limb arteries. Here, we investigated if Lp(a) plays a specific role in ED due with symptomatic pelvic artery atherosclerosis. Patients and methods: Out of 276 consecutive patients treated for ED with angioplasties on proximal (69%) and distal (31%, distal to Alcock channel) erection-related arteries, 236 patients (age: 62±10 years) of which Lp(a) values were available were retrospectively analyzed. Results: The baseline International Index of Erectile Function-15 (IIEF-15) score was 29±15 and significantly increased to 43±20 (increase: 14±21) after treatment at average follow up of 286±201 days. In 25%, Lp(a) values were elevated to more than 30 mg/dL. Hypercholesterolemia, coronary, lower extremity peripheral, and polyvascular disease were more common in patients with Lp(a) ≥60 mg/dl. Anatomic arterial lesion distribution (proximal/distal), improvement in IIEF-15 and clinically driven re-intervention rate (overall 7%) did not differ between patients with <30, 30-59, and ≥60 mg/dL Lp(a). Conclusions: While angioplasty is an effective therapy for ED of arterial origin in patients with obstruction of erection-related arteries, Lp(a) does not seem to play a major role for clinical outcomes in these patients.

Prospective clinical study testing the efficacy and safety of a new formula to increase the precision of oxygen therapy in the initiation phase of cardiopulmonary bypass.

INTRODUCTION: During cardiopulmonary bypass (CPB), supranormal concentrations of oxygen are routinely administered with the intention to prevent cellular hypoxia. However, hyperoxemia may have adverse effects on patient outcome. Oxygen settings are based on the perfusionist's individual work experience rather than profound recommendations and studies analyzing the effect of oxygen levels are in need of methodological improvement. We aimed to advance perfusion technique by developing and clinically applying a formula for tailored oxygen therapy in CPB. METHODS: A formula to precalculate the oxygenator setting before CPB was developed. The newly-derived formula was then evaluated in a prospective, single-center pilot study to test whether a predefined arterial partial oxygen pressure (PaO2) of 150-250 mmHg could be reached. 80 patients were enrolled in the study between April and September 2021. RESULTS: The mean oxygen fraction calculated for the setting of the gas blender was 52% ±0,12. The mean PaO2 after initiation of the CPB was 193 ± 99 mmHg (min-max: 61-484, median 163 mmHg). 38.75% of the values were in the desired PaO2 corridor of 150 to 250 mmHg. 8.75% of all PaO2 values were below <79.9 mmHg, 31.25% between 80 and 149.9 mmHg, 38.75% between 150 and 249.9 mmHg and 21.25%>250 mmHg. CONCLUSIONS: Conceptually, perfusion technique should be goal-directed, guided by objective parameters and formulas. Although the optimal CPB oxygenation target remains unknown, it is nevertheless important to develop strategies to tailor oxygen therapy to aid in creating evidence as to what level of oxygen is best for patients during CPB. The formula we derived needs further adjustments to increase results in the target range.

A novel heat treatment protocol for human milk.

Human milk (HM) is the recommended nutrition for premature infants, but it may require processing to ensure microbial safety. The current standard is Holder pasteurisation (HoP), i.e. heating milk at 62.5 ± 0.5°C for 30 min, which eliminates bacteria but destroys heat labile bioactive HM components. We aimed to test an alternative thermal method, high-temperature short-time (HTST) pasteurisation using a modified Holder pasteurisation platform as this method has shown to preserve proteins in experimental HM flow pasteurisers. We analysed the ability of this batch process to eliminate bacterial species and to retain alkaline phosphatase, secretory immunoglobulin A and lactoferrin in HM. HTST at 81°C/5 s was as effective as HoP in bacterial count reduction while the retention of bioactive components was only improved at 62°C/5 s as compared to 72°C/5 s and HoP. HTST is a promising alternative to HoP but an optimal temperature-time combination needs to be determined for each technical platform separately.

Cytokine Adsorber Use during DCD Heart Perfusion Counteracts Coronary Microvascular Dysfunction.

Microvascular dysfunction (MVD) in cardiac allografts is associated with an impaired endothelial function in the coronary microvasculature. Ischemia/reperfusion injury (IRI) deteriorates endothelial function. Hearts donated after circulatory death (DCD) are exposed to warm ischemia before initiating ex vivo blood perfusion (BP). The impact of cytokine adsorption during BP to prevent MVD in DCD hearts is unknown. In a porcine DCD model, we assessed the microvascular function of hearts after BP with (DCD-BPCytoS, n = 5) or without (DCD-BP, n = 5) cytokine adsorption (CytoSorb®). Microvascular autoregulation was assessed by increasing the coronary perfusion pressure, while myocardial microcirculation was measured by Laser-Doppler-Perfusion (LDP). We analyzed the immunoreactivity of arteriolar oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal (HNE), endothelial injury indicating cell adhesion molecules CD54, CD106 and CD31, and eNOS. We profiled the concentration of 13 cytokines in the perfusate. The expression of 84 genes was determined and analyzed using machine learning and decision trees. Non-DCD hearts served as a control for the gene expression analysis. Compared to DCD-BP, relative LDP was improved in the DCD-BPCytoS group (1.51 ± 0.17 vs. 1.08 ± 0.17). Several pro- and anti-inflammatory cytokines were reduced in the DCD-BPCytoS group. The expression of eNOS significantly increased, and the expression of nitrotyrosine, HNE, CD54, CD106, and CD31, markers of endothelial injury, majorly decreased in the DCD-BPCytoS group. Three genes allowed exact differentiation between groups; regulation of HIF1A enabled differentiation between perfusion (DCD-BP, DCD-BPCytoS) and non-perfusion groups. CAV1 allowed differentiation between BP and BPCytoS. The use of a cytokine adsorption device during BP counteracts preload-dependent MVD and preserves the microvascular endothelium by preventing oxidative stress and IRI of coronary arterioles of DCD hearts.

Prediction Model for Contractile Function of Circulatory Death Donor Hearts Based on Microvascular Flow Shifts During Ex Situ Hypothermic Cardioplegic Machine Perfusion.

Background Hearts procured from circulatory death donors (DCD) are predominantly maintained by machine perfusion (MP) with normothermic donor blood. Currently, DCD heart function is evaluated by lactate and visual inspection. We have shown that MP with the cardioplegic, crystalloid Custodiol-N solution is superior to blood perfusion to maintain porcine DCD hearts. However, no method has been developed yet to predict the contractility of DCD hearts after cardioplegic MP. We hypothesize that the shift of microvascular flow during continuous MP with a cardioplegic preservation solution predicts the contractility of DCD hearts. Methods and Results In a pig model, DCD hearts were harvested and maintained by MP with hypothermic, oxygenated Custodiol-N for 4 hours while myocardial microvascular flow was measured by Laser Doppler Flow (LDF) technology. Subsequently, hearts were perfused with blood for 2 hours, and left ventricular contractility was measured after 30 and 120 minutes. Various novel parameters which represent the LDF shift were computed. We used 2 combined LDF shift parameters to identify bivariate prediction models. Using the new prediction models based on LDF shifts, highest r2 for end-systolic pressure was 0.77 (P=0.027), for maximal slope of pressure increment was 0.73 (P=0.037), and for maximal slope of pressure decrement was 0.75 (P=0.032) after 30 minutes of reperfusion. After 120 minutes of reperfusion, highest r2 for end-systolic pressure was 0.81 (P=0.016), for maximal slope of pressure increment was 0.90 (P=0.004), and for maximal slope of pressure decrement was 0.58 (P=0.115). Identical prediction models were identified for maximal slope of pressure increment and for maximal slope of pressure decrement at both time points. Lactate remained constant and therefore was unsuitable for prediction. Conclusions Contractility of DCD hearts after continuous MP with a cardioplegic preservation solution can be predicted by the shift of LDF during MP.

Holder Pasteurization: Comparison of Water-Bath and Dry-Tempering Devices.

Background: Human milk (HM) for premature infants is frequently Holder pasteurized (heated at 62.5 ± 0.5°C for 30 min) despite its detrimental effects on heat-sensitive milk components. This tolerated compromise ensures HM's microbial safety while less detrimental methods like short-time HM treatments (HTST) are still being evaluated. Dry-tempering devices (DT-HoP) were recently introduced in clinical practice due to hygienic concerns about water-based Holder pasteurizers (WB-HoP). Evidence on the impact of such dry-tempering devices on HM quality is lacking. The aim of this study was to compare protein retention rates after DT-HoP, WB-HoP and HTST. Methods: We colorimetrically determined alkaline phosphatase activity (ALP), concentrations of secretory immunoglobulin A (sIgA), and lactoferrin (LF) before and after DT-HoP, WB-HoP and HTST. Results: ALP was below the detection limit after HoP, but retained 52.8 ± 13% activity after HTST (p < 0.01). Secretory IgA (WB-HoP = 73.2 ± 13.5% vs. DT-HoP = 57 ± 14%, p = 0.0018) and LF retention (WB-HoP=47 ± 40% vs. DT-HoP=25 ± 8%, p = 0.07) differed between the two HoP modes. Again, retention was better maintained after HTST compared to HoP (80.4 ± 23% sIgA and 70 ± 42% LF concentration, all p < 0.01). Conclusion: Dry-tempering milk lowers even further the quality of HM when performing HoP compared to water-bath pasteurization, while HTST warrants continued evaluation for clinical application.

Genotyping of familial Mediterranean fever gene (MEFV)-Single nucleotide polymorphism-Comparison of Nanopore with conventional Sanger sequencing.

BACKGROUND: Through continuous innovation and improvement, Nanopore sequencing has become a powerful technology. Because of its fast processing time, low cost, and ability to generate long reads, this sequencing technique would be particularly suitable for clinical diagnostics. However, its raw data accuracy is inferior in contrast to other sequencing technologies. This constraint still results in limited use of Nanopore sequencing in the field of clinical diagnostics and requires further validation and IVD certification. METHODS: We evaluated the performance of latest Nanopore sequencing in combination with a dedicated data-analysis pipeline for single nucleotide polymorphism (SNP) genotyping of the familial Mediterranean fever gene (MEFV) by amplicon sequencing of 47 clinical samples. Mutations in MEFV are associated with Mediterranean fever, a hereditary periodic fever syndrome. Conventional Sanger sequencing, which is commonly applied in clinical genetic diagnostics, was used as a reference method. RESULTS: Nanopore sequencing enabled the sequencing of 10 target regions within MEFV with high read depth (median read depth 7565x) in all samples and identified a total of 435 SNPs in the whole sample collective, of which 29 were unique. Comparison of both sequencing workflows showed a near perfect agreement with no false negative calls. Precision, Recall, and F1-Score of the Nanopore sequencing workflow were > 0.99, respectively. CONCLUSIONS: These results demonstrated the great potential of current Nanopore sequencing for application in clinical diagnostics, at least for SNP genotyping by amplicon sequencing. Other more complex applications, especially structural variant identification, require further in-depth clinical validation.

A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test.

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

Serological and viral genetic features of patients with COVID-19 in a selected German patient cohort-correlation with disease characteristics.

To study host-virus interactions after SARS coronavirus-2 (SARS-CoV-2) infection, genetic virus characteristics and the ensued humoral immune response were investigated for the first time. Fifty-five SARS-CoV-2-infected patients from the early pandemic phase were followed up including serological testing and whole genome sequencing. Anti-spike and nucleocapsid protein (S/N) IgG and IgM levels were determined by screening ELISA and IgG was further characterized by reactivity to S-subunit 1 (anti-S1), S-subunit 2 (anti-S2) and anti-N. In 55 patients, 90 genetic SARS-CoV-2 changes including 48 non-synonymous single nucleotide variants were identified. Phylogenetic analysis of the sequencing data showed a cluster representing a local outbreak and various family clusters. Anti-S/N and anti-N IgG were detected in 49 patients at an average of 83 days after blood collection. Anti-S/N IgM occurred significantly less frequently than IgG whereas anti-S2 was the least prevalent IgG reactivity (P < 0.05, respectively). Age and overweight were significantly associated with higher anti-S/N and anti-S1 IgG levels while age only with anti-N IgG (multiple regression, P < 0.05, respectively). Anti-S/N IgG/IgM levels, blood group A + , cardiovascular and tumour disease, NSP12 Q444H and ORF3a S177I were independent predictors of clinical characteristics with anti-S/N IgM being associated with the need for hospitalization (multivariate regression, P < 0.05, respectively). Anti-SARS-CoV-2 antibody generation was mainly affected by higher age and overweight in the present cohort. COVID-19 traits were associated with genetic SARS-CoV-2 variants, anti-S/N IgG/IgM levels, blood group A + and concomitant disease. Anti-S/N IgM was the only antibody associated with the need for hospitalization.

Relationship of Laser-Doppler-Flow and coronary perfusion and a concise update on the importance of coronary microcirculation in donor heart machine perfusion.

BACKGROUND: Machine perfusion (MP) is a novel method for donor heart preservation. The coronary microvascular function is important for the transplantation outcome. However, current research on MP in heart transplantation focuses mainly on contractile function. OBJECTIVE: We aim to present the application of Laser-Doppler-Flowmetry to investigate coronary microvascular function during MP. Furthermore, we will discuss the importance of microcirculation monitoring for perfusion-associated studies in HTx research. METHODS: Porcine hearts were cardioplegically arrested and harvested (Control group, N = 4). In an ischemia group (N = 5), we induced global ischemia of the animal by the termination of mechanical ventilation before harvesting. All hearts were mounted on an MP system for blood perfusion. After 90 minutes, we evaluated the effect of coronary perfusion pressures from 20 to 100 mmHg while coronary laser-doppler-flow (LDF) was measured. RESULTS: Ischemic hearts showed a significantly decreased relative LDF compared to control hearts (1.07±0.06 vs. 1.47±0.15; p = 0.034). In the control group, the coronary flow was significantly lower at 100 mmHg of perfusion pressure than in the ischemia group (895±66 ml vs. 1112±32 ml; p = 0.016). CONCLUSIONS: Laser-Doppler-Flowmetry is able to reveal coronary microvascular dysfunction during machine perfusion of hearts and is therefore of substantial interest for perfusion-associated research in heart transplantation.

Reconditioning of circulatory death hearts by ex-vivo machine perfusion with a novel HTK-N preservation solution.

BACKGROUND: Warm ischemia followed by blood reperfusion is associated with reduced myocardial contractility. Circulatory death (CD) hearts are maintained by machine perfusion (MP) with blood. However, the impact of MP with histidine-tryptophane-ketoglutarate (HTK) or novel HTK-N solution on reconditioning of CD-heart contractility is unknown. METHODS: In a porcine model, native hearts were directly harvested (control), or CD was induced before harvesting, followed by left ventricular (LV) contractile assessment. In MP-groups, CD-hearts were maintained for 4 h by MP with blood (CD-B), cold oxygenated HTK (CD-HTK) or HTK-N (CD-HTK-N) before contractile evaluation (all groups n = 8). We performed immunohistochemistry of LV myocardial samples. We profiled myocardial expression of 84 oxidative stress-related genes and correlated the findings with myocardial contractility via a machine learning algorithm. RESULTS: HTK-N improved end-systolic pressure (ESP=172±10 vs 132±5 mmHg, p = 0.02) and maximal slope of pressure increment (dp/dtmax=2161±214 vs 1240±167 mmHg/s, p = 0.005) compared to CD, whereas CD-B failed to improve contractility. Dp/dtmax (2161±214 vs 1177±156, p = 0.08) and maximal rate of pressure decrement (dp/dtmin=-1501±228 vs -637±79, p = 0.005) were also superior in CD-HTK-N compared to CD-B. In CD-HTK-N, myocardial 4-hydroxynonenal (marker for oxidative stress; p<0.001), nitrotyrosine (marker for nitrosative stress; p = 0.004), poly(adenosine diphosphate-ribose)polymerase (marker for necrosis; p = 0.028) immunoreactivity and cell swelling (p = 0.008) were decreased compared to CD-B. Strong correlation of gene expression with ESP was identified for oxidative stress defense genes in CD-HTK-N. CONCLUSION: During harvesting procedure, MP with HTK-N reconditions CD-heart systolic and diastolic function by reducing oxidative and nitrosative stress and preventing cardiomyocytes from cell swelling and necrosis.

Impact of skeletonized harvesting of the internal thoracic artery on intrasternal microcirculation considering preparation quality.

OBJECTIVES: Previous studies have demonstrated the impact of internal thoracic artery (ITA) harvesting on microcirculation in parasternal tissues. However, the impact of skeletonized ITA harvesting on intrasternal microcirculation is unknown. Intraskeletal tissue perfusion has been proven to be crucial for deep wound healing. Furthermore, the impact of different levels of surgical preparation quality on intrasternal microcirculation has not been investigated yet. METHODS: Sternal microcirculation (sLDP) was monitored with a novel Laser Doppler Perfusion needle probe, while the ITA was skeletonized in a pig model. To mimic different levels of preparation quality, satellite veins were either coagulated or not during preparation. To show the effect of ideally avoiding any surgical manipulation on sLDP, the ITA was clipped in a third sham-harvested group. RESULTS: sLDP was reduced highly significant to 71 [standard deviation (SD): 9]% (P < 0.001) after skeletonized harvesting of the ITA. Coagulation of the satellite veins as a detrimental surgical factor resulted in a significantly stronger reduction of sLDP to 56 (SD: 11)% (P < 0.05) compared to sLDP with non-coagulated satellite veins. ITA clipping reduced sLDP highly significant to 71 (SD: 8)% (P < 0.001) in the sham-operated group. CONCLUSIONS: ITA harvesting markedly impairs microcirculation of the sternum but remains unavoidable when coronary artery bypass grafting should be performed. Nevertheless, excessive surgical damage and coagulation of satellite veins is avoidable and should be reduced to a minimum to minimize the risk of deep sternal wound healing complications.

Label-free identification and differentiation of different microplastics using phasor analysis of fluorescence lifetime imaging microscopy (FLIM)-generated data.

As plastic pollution is becoming an increasing worldwide problem, a variety of different techniques for the detection and in-depth characterization of plastics, including spectroscopy and chromatography methods, were introduced to the public. Recently we presented fluorescence lifetime imaging microscopy (FLIM) a new approach for the identification and characterization of microplastics using their fluorescence lifetime (τ) for differentiation. A very powerful extension of the recently established FLIM could be phasor analysis, which allows data representation in an interactive 2D graphical phasor plot thereby enabling a global view of the fluorescence decay in each pixel of the measured image. Microplastic particles generated from six different types of plastics were subjected to excitation wavelengths of 440 nm, upon which specific fluorescence lifetimes as well as the photon yield were determined using FLIM and phasor analysis. We could show that phasor analysis for FLIM with a laser pulse repetition frequency of 40 MHz was able to generate specific locations in the phasor plot for the plastics for fast differentiation, e.g. resulting in well-defined phasor plot positions for ABS at 3.019 ns, PPE at 6.239 ns, PET bottle from Germany at 2.703 ns and PET bottle from USA at 2.711 ns. Phasor analysis for FLIM proves to be a fast, label-free, and sensitive method for the identification and differentiation of plastics also with the aid of visualization variation enabling techniques such as heat treatment of plastics.