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 offenses and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from nonathletes were analyzed 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 nonapproved proliferator-activated receptor delta agonist GW1516) in a total of 81 samples, with 91% of these suspected cases being verified by the confirmation method. In addition to 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.

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 .

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.

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.

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.

SARS-CoV-2 antibody testing-questions to be asked.

Severe acute respiratory syndrome coronavirus 2 infection and development of coronavirus disease 2019 presents a major health care challenge of global dimensions. Laboratory diagnostics of infected patients, and the assessment of immunity against severe acute respiratory syndrome coronavirus 2, presents a major cornerstone in handling the pandemic. Currently, there is an increase in demand for antibody testing and a large number of tests are already marketed or are in the late stage of development. However, the interpretation of test results depends on many variables and factors, including sensitivity, specificity, potential cross-reactivity and cross-protectivity, the diagnostic value of antibodies of different isotypes, and the use of antibody testing in identification of acutely ill patients or in epidemiological settings. In this article, the recently established COVID-19 Task Force of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) addresses these issues on the basis of currently available data sets in this rapidly moving field.

Nanopore Sequencing in a Clinical Routine Laboratory: Challenges and Opportunities.

BACKGROUND: About forty-five years ago the advent of Sanger sequencing (Sanger and Coulson 1975) was revolutionary as it allowed deciphering of complete genome sequences. A second revolution came when next-generation sequencing (NGS) technologies accelerated and cheapened genome sequencing. Recently, third generation/longread sequencing methods have appeared, which can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. Nanopore sequencing is one of these third-generation approaches, enabling a single molecule of DNA or RNA to be sequenced in real-time without the need for PCR amplification or chemical labelling of the sample. It works by monitoring changes to an electrical current as nucleic acids are passed through protein or synthetic nanopores. METHODS: A literature search was performed in order to collect and summarize current information about the methodological aspects of nanopore sequencing as well as some application examples. RESULTS: The review describes concisely and comprehensibly the technical aspects of nanopore sequencing and stresses the advantages and disadvantages of this technique thereby also giving examples of their potential applications in the clinical routine laboratory as are rapid identification of viral pathogens, monitoring Ebola, environmental and food safety monitoring, human and plant genome sequencing, monitoring of antibiotic resistance, and other applications. CONCLUSIONS: It is a useful incitation for such ones being permanently in search of upgrading their laboratory.

First Results from a Screening of 300 Naturally Occurring Compounds: 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol, 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol, and 5-epi-nakijinone Q as Substances with the Potential for Anticancer Therapy.

There is a variety of antineoplastic drugs that are based on natural compounds from ecological niches with high evolutionary pressure. We used two cell lines (Jurkat J16 and Ramos) in a screening to assess 300 different naturally occurring compounds with regard to their antineoplastic activity. The results of the compounds 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol (P01F03), 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol (P01F08), and 5-epi-nakijinone Q (P03F03) prompted us to perform further research. Using viability and apoptosis assays on the cell lines of primary human leukemic and normal hematopoietic cells, we found that P01F08 induced apoptosis in the cell lines at IC50 values between 1.61 and 2.95 µM after 72 h. IC50 values of peripheral blood mononuclear cells (PBMNCs) from healthy donors were higher, demonstrating that the cytotoxicity in the cell lines reached 50%, while normal PBMNCs were hardly affected. The colony-forming unit assay showed that the hematopoietic progenitor cells were not significantly affected in their growth by P01F08 at a concentration of 3 µM. P01F08 showed a 3.2-fold lower IC50 value in primary leukemic cells [acute myeloid leukemia (AML)] compared to the PBMNC of healthy donors. We could confirm the antineoplastic effect of 5-epi-nakijinone Q (P03F03) on the cell lines via the induction of apoptosis but noted a similarly strong cytotoxic effect on normal PBMNCs.

Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients.

Circulating tumor cells (CTCs) are promising biomarkers for diagnosis and therapy in systemic cancer. However, their infrequent and unreliable detection, especially in nonmetastatic cancer, currently impedes the clinical use of CTCs. Because leukapheresis (LA) targets peripheral blood mononuclear cells, which have a similar density to CTCs, and usually involves processing the whole circulating blood, we tested whether LA could substantially increase CTC detection in operable cancer patients. Therefore, we screened LA products generated from up to 25 L of blood per patient in two independent studies, and found that CTCs can be detected in more than 90% of nonmetastatic breast cancer patients. Interestingly, complete white blood cell sampling enabled determining an upper level for total CTC numbers of about 100,000 cells (median, 7,500 CTCs) per patient and identified a correlation of CTC numbers with anatomic disease spread. We further show that diagnostic leukapheresis can be easily combined with the US Food and Drug Administration-approved CellSearch system for standardized enumeration of CTCs. Direct comparison with 7.5 mL of blood revealed a significantly higher CTC frequency in matched LA samples. Finally, genomic single-cell profiling disclosed highly aberrant CTCs as therapy-escaping variants in breast cancer. In conclusion, LA is a clinically safe method that enabled a reliable detection of CTCs at high frequency even in nonmetastatic cancer patients, and might facilitate the routine clinical use of CTCs as in the sense of a liquid biopsy. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring of early systemic cancer.

Effects of radiographic contrast media on the micromorphology of the junctional complex of erythrocytes visualized by immunocytology.

Effects of radiographic contrast media (RCM) application were demonstrated in vitro and in vivo where the injection of RCM into the A. axillaris of patients with coronary artery disease was followed by a significant and RCM-dependent decrease of erythrocyte velocity in downstream skin capillaries. Another study in pigs revealed that the deceleration of erythrocytes coincided with a significant reduction of the oxygen partial pressure in the myocardium--supplied by the left coronary artery--after the administration of RCM into this artery. Further reports showed RCM dependent alterations of erythrocytes like echinocyte formation and exocytosis, sequestration of actin or band 3 and the buckling of endothelial cells coinciding with a formation of interendothelial fenestrations leading to areas devoid of endothelial cells. Key to morphological alterations of erythrocytes is the membrane cytoskeleton, which is linked to the band 3 in the erythrocyte membrane via the junctional complex. Fundamental observations regarding the cell biological and biochemical aspects of the structure and function of the cell membrane and the membrane cytoskeleton of erythrocytes have been reported. This review focuses on recent results gained, e.g., by advanced confocal laser scanning microscopy of different double-stained structural elements of the erythrocyte membrane cytoskeleton.

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.

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.

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.

Storage-induced changes of the cytosolic red blood cell proteome analyzed by 2D DIGE and high-resolution/high-accuracy MS.

The storage of packed red blood cells (RBCs) is associated with the development of morphological and biochemical changes leading to a reduced posttransfusion functionality and viability of the cells. Within this study, 2D DIGE and high-resolution/high-accuracy Orbitrap MS were used to analyze the storage-induced changes of the cytosolic RBC proteome and identify characteristic protein patterns and potential marker proteins for the assessment of RBC storage lesions. Leukodepleted RBC concentrates were stored according to standard blood bank conditions for 0, 7, 14, 28, and 42 days and analyzed by using a characterized and validated protocol. Following statistical evaluation, a total of 14 protein spots were found to be significantly altered after 42 days of ex vivo storage. Protein identification was accomplished by tryptic digestion and LC-MS/MS and three proteins potentially useful as biomarkers for RBC aging comprising transglutaminase 2, beta actin, and copper chaperone for superoxide dismutase were selected and validated by western blot analysis. These can serve as a basis for the development of a screening assay to detect RBC storage lesions and autologous blood doping in sports.

Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS.

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome.

Platelet transfusion alters CD40L blood level and release capacity in patients suffering from thrombocytopenia.

BACKGROUND: Platelet (PLT)-derived cytokines, such as soluble CD40 ligand (sCD40L), play an important role in the development of adverse transfusion reactions associated with the administration of PLT products. In this study, we determined sCD40L concentration and release capacity in patients with thrombocytopenia before and after receiving a PLT transfusion. STUDY DESIGN AND METHODS: The study included 12 patients suffering from chemotherapy-induced thrombocytopenia. sCD40L levels and release capacity were measured in plasma samples of the patients before and after PLT administration as well as in the respective plateletpheresis concentrates by enzyme-linked immunosorbent assay. Sixteen healthy blood donors served as a control group. RESULTS: In PLT concentrates, elevated sCD40L levels (2567±134 pg/mL) were observed in comparison to plasma sCD40L levels in controls (238.4±35.3 pg/mL). sCD40L plasma concentration of patients with thrombocytopenia was significantly reduced (86.3±16.7 pg/mL) before transfusion and increased to nearly normal levels (204.4±24.8 pg/mL) after PLT administration. In parallel, the sCD40L release capacity per PLT showed no significant difference between controls and patients with thrombocytopenia before transfusion (33.3±2.6 and 29.3±4.6 ag/PLT, respectively) but was significantly reduced after PLT transfusion (22.4±2.7 compared to 29.3±4.6 ag/PLT). CONCLUSIONS: In patients with thrombocytopenia, sCD40L levels were clearly influenced by PLT transfusions: PLT administration led to a normalization of sCD40L plasma concentration. Nevertheless, adverse transfusion reactions did not occur in these patients. The sCD40L release capacity was enhanced by PLT administration dependent on the increase in the amount of PLT count.

Determination of thromboxane formation, soluble CD40L release and thrombopoietin clearance in apheresis platelet concentrates.

All deleterious changes in platelet morphology, structure and function that occur in platelet concentrates (PC) during storage are titled as the 'platelet storage lesion'. No single in vitro test currently available is sufficient in assessing these changes of platelet quality. The release of soluble CD40 Ligand (sCD40L), the formation of thromboxane (TXB2) and the thrombopoietin (TPO) clearance reflect different aspects of platelet metabolism and activitiy, and were used to examine platelet quality in apheresis platelet products. At days 1, 3 and 5, in single-donor apheresis platelet products (n = 10) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). TPO (measured by ELISA) was determined after an incubation time of 5 h at 37°C with platelet-rich plasma (adjusted initial TPO concentration of about 500 pg/mL). Results were related to a therapeutic unit (TU = 2 × 10(11) platelets). Immediately after platelet preparation, sCD40L release was 41 ± 7.6 ng/TU, TXB2 formation 1688 ± 374 ng/TU and TPO clearance 1.22 ± 0.32 ng/h/TU. At days 1, 3 and 5, sCD40L was reduced to 89 ± 7%, 71 ± 12% and 57 ± 9%, TXB2 release to 91 ± 6%, 74 ± 12% and 58 ± 9% and TPO clearance to 90 ± 15%, 84 ± 5% and 79 ± 10% of the respective control values. In conclusion, in single-donor apheresis PC, sCD40L release and TXB2 formation as well as TPO clearance by the platelets were dependent on storage duration and reduced to about 60% to 80% of the respective control values after a storage period for 5 days. These findings are in line with literature data, indicating that a loss of platelet functionality of about 30% will occur after 5 days of storage.

Soluble CD40L release as test for functional platelet loss.

BACKGROUND: During platelet storage, alterations of the platelet function, 'platelet storage lesion', can be observed resulting in a reduced platelet viability. The release of soluble CD40 ligand (sCD40L) by platelets reflects different aspects of platelet metabolism and activity. Therefore, we used the sCD40L release to test for functional platelet loss in platelet products during storage in comparison to the formation of thromboxane (TXB2). METHODS: On day 1, 3, and 5 in single donor apheresis platelet products (n = 8) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). Results were related to a therapeutic unit (TU = 2 x 10*11 platelets). RESULTS: In platelet-rich plasma of the donors, sCD40L release was 42.5 +/- 7.1 ng/TU and TXB2 formation 2,183 +/- 576 ng/TU. On day 1, 3, and 5 sCD40L release was reduced to 95%, 64%, and 57% and TXB2 formation to 92%, 80%, and 65% of the respective control values. CONCLUSIONS: In single donor apheresis PCs, sCD40L release and TXB2 formation showed a comparable course over storage time and were reduced to about 60% of the respective control values after a storage period of 5 days. These findings are in line with literature data indicating that a functional platelet loss of about 30% will occur after 5 days of storage. Overall, sCD40L release could be easily induced by recalcification and clot formation and can be used as a marker for functional platelet loss.

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.

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.