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Item type: Item , A Validation Approach for Determining Fetal Blood Groups Non-Invasively by High-Sensitive Next-Generation Sequencing(2025) Wienzek-Lischka, Sandra; Soelter, Marion; Froelich, Annika; Ernst-Schlegel, Marion; Gattenloehner, Stefan; Braeuninger, Andreas; Sachs, Ulrich J.Introduction: For pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the fetus and newborn (HDFN), prenatal intervention in subsequent pregnancies may be necessary to prevent complications for the fetus. A non-invasive prenatal diagnostic procedure (NIPD) is recommended for fetal blood group genotyping. RT-PCR is used for fetal RHD determination as a reliable screening method with high sensitivity and specificity. For other antigens with variants involving single-base substitutions, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) are recommended to reduce the risk of false-negative results. Only NGS offers the possibility of determining the cell-free fetal DNA (cffDNA) fraction in maternal plasma by sequencing additional gene fragments in parallel, but no standard exists for assay validation. Material and Methods: A custom-made primer panel was designed to target the common platelet and red cell antigens involved in fetal red cell and platelet incompatibilities, as well as additional anonymous single-nucleotide polymorphism (SNP) targets for use as an internal control. Amplicon-based NGS was carried out using semiconductor sequencing. For HPA-1a (HPA*1A, ITGB3) and K (KEL*01.01, KEL) assay validation, the limit of detection (LOD) and limit of quantification (LOQ) were estimated, as were false-positive antithetic alleles, linearity, and inter-assay variation, using cell-free DNA (cfDNA) extracted from the blood samples of healthy blood donors. An additional analysis was performed using 23 diagnostic samples from 21 pregnant women. Results: Regression analysis of dilution series using HPA-1a- and K-positive cell-free plasma samples in antigen-negative donor plasma showed that recovery is definitely feasible up to an HPA*1A and KEL*01.01 allele frequency of 1%. Base calls of false-positive antithetic alleles were detected with a maximum of 0.25% using 21 healthy blood donors. The LOD was estimated to be 0.2057% (mean + 3 SD) for HPA*1A with a LOQ of 0.6298% (mean + 10 SD). For KEL*01.01, the LOD was 0.1706% (mean + 3 SD) and the LOQ was 0.5314% (mean + 10 SD). The analysis of 15 of 21 cases with diagnostic samples from pregnant women with neonatal blood available for confirmatory testing resulted in 100% concordant results. The fetal fraction of these samples was calculated with a median of 11.03% (95% CI: 8.89, 13.20). Conclusions: NGS for non-invasive fetal blood group genotyping is an accurate and reliable method. In-house validation of the used assays can be performed using healthy donors to determine the LOD, LOQ and sensitivity. The threshold for paternally inherited fetal HPA*1A and KEL*01.01 alleles could be set at 1% (i.e., 2% fetal fraction) to obtain reliable test results. Internal controls for assessing the fetal fraction are essential to avoid false-negative test results.Item type: Item , Comparative Assessment of Sperm Morphology in Liquid-Preserved Boar Semen Using Cytological Stains(2025) Braune, Annika; Wehrend, Axel; Kauffold, Johannes; Farshad, AbbasAccurate assessment of sperm morphology is essential for artificial insemination using liquid-preserved boar semen. This study compared nine commonly used staining techniques, eosin, eosin–nigrosin, Diff-Quick®, Hemacolor®, Sangodiff-G®, Spermac®, Formol–Citrate–Rose Bengal stain, Testsimplets®, and Methyl Violet, based on morphological assessment, cost, time efficiency, and storage stability. Each staining method was applied to 36 slides, totaling 324 samples, and evaluated four times each (1296 evaluations). Slides were analyzed four times: immediately after staining and after 1 day, 1 week, and 3 months of storage. The results indicated that Eosin was the fastest and most cost-effective method, providing strong contrast, though it increased detection of structural alterations. Eosin–nigrosin offered detailed morphology but formed colored crystals over time. Diff-Quick® and Hemacolor® showed good initial performance, but Hemacolor® lost pigment clarity after 3 months (p = 0.0273). Sangodiff-G® had poor contrast and reduced detection of abnormalities (p = 0.00229). Spermac® delivered high contrast but was time-consuming. Formol–Citrate–Rose Bengal stain required extensive preparation and showed significant post-storage changes (p < 0.0001). Testsimplets®, despite their high cost, suffered from declining interpretability (p < 0.0001). Methyl Violet lacked sufficient resolution and was highly unstable over time (p < 0.0001). In conclusion, Eosin emerged as the most practical and economical staining method for routine morphological evaluation of liquid-preserved boar semen. While eosin–nigrosin was also effective, its storage instability limits broader application. Other methods showed specific weaknesses, emphasizing the need to tailor stain selection to laboratory goals and constraints.Item type: Item , Implementing Culture of Care in Germany(2025) Ameli, Katharina; Krämer, StephanieThe implementation of the 3Rs principles is an essential part of daily routines and structural processes in animal research. The work of Russell and Burch regarding the 3Rs has been implemented in the field over the course of decades, but since 2002, the concept of a Culture of Care has come more to the forefront. In the present project, 503 experts in Germany were exploratively surveyed about their individual perceptions of Culture of Care and its implementation within their institutions. Using a questionnaire with closed questions (five-point agreement scale) and open questions, the data offer insights into Culture of Care in Germany. The results allow for the initial conclusion that a Culture of Care has not been fully established yet. Further research is needed to address a holistic and critical interdisciplinary Culture of Care that focuses on the animals’ perspective in a transformative way.Item type: Item , Evaluating the Safety and Efficacy of Intravenous Thrombolysis in Acute Ischemic Stroke Patients Without Perfusion Deficit: A Retrospective Analysis(2025) Alhaj Omar, Omar; Gerner, Stefan T.; Alikevitch, Slava; Hamzic, Samra; Viard, Maxime; Mrochen, Anne; Böttger, Priyanka; Juenemann, Martin; Braun, TobiasBackground/Objectives: Acute ischemic stroke (AIS) remains a major cause of morbidity and mortality worldwide. Although advanced imaging modalities, such as CT perfusion (CTP), are increasingly being used in clinical decision-making, the necessity and added value of perfusion imaging prior to intravenous thrombolysis (IVT) within early time windows remains uncertain. We aim to evaluate the safety and functional outcomes of IVT in AIS patients without perfusion deficits on CTP. We question the requirement of perfusion mismatch for IVT eligibility and hypothesize that IVT is safe and beneficial even in the absence of a perfusion deficit. Methods: A retrospective analysis was conducted using data from the Giessen Stroke Registry, focusing on AIS patients who underwent CTP imaging and received IVT between 01/2018 and 12/2020. Patients who underwent endovascular therapy were excluded. Clinical data, including demographics, National Institutes of Health Stroke Scale (NIHSS) scores, modified Rankin Scale (mRS) scores, and complications, were collected. Patients were dichotomized based on the presence of perfusion lesions and compared in terms of efficacy outcomes (i.e., NIHSS or mRS improvement during the hospital stay) and safety outcomes (i.e., post-thrombolytic hemorrhagic complications). Results: Of the 89 AIS patients with available CTP data who received IVT, 34 (38%) had a perfusion deficit and 55 (62%) did not. There were no significant differences between the groups in terms of hemorrhagic complications or functional outcomes at discharge (NIHSS and mRS). Clinical improvement from admission to discharge was similar in both groups. Conclusions: Our findings suggest that IVT is safe and clinically effective even in AIS patients without detectable perfusion deficits on CTP within the standard therapeutic window. These results support current guideline recommendations that do not mandate perfusion imaging for early presenters. Routine use of CTP in this context may be of limited clinical utility and could unnecessarily delay treatment or introduce additional risks in the first 4.5 h.Item type: Item , In Vitro Inhibition of Cryptosporidium parvum Infection by the Olive Oil Component Oleocanthal(2025) Ampama, M. Nguele; Hanke, Dominik; Velásquez, Zahady D.; Wäber, Nadine B.; Hermosilla, Carlos; Taubert, Anja; Mazurek, SybilleHuman cryptosporidiosis caused by the zoonotic apicomplexan parasite Cryptosporidium parvum represents a neglected and re-emerging poverty-related disease. C. parvum possesses minimalistic metabolic capacities and highly depends on its intestinal epithelial host cell for intracellular replication. Based on previous results showing that glycolysis and glutaminolysis inhibition diminished C. parvum replication in vitro, we here investigated the impact of the olive oil component oleocanthal on C. parvum infection in HCT-8 cells under physioxia (5% O2) and hyperoxia (21% O2). Oleocanthal targets a broad spectrum of regulatory molecules, amongst which mTOR represents a master regulator of glycolysis and glutaminolysis. Using a host cell pre-treatment as well as a pre- and post-infection treatment protocol, 5 µM oleocanthal reduced C. parvum infection rates between 51% and 94%. Host cellular metabolic conversion rates linked oleocanthal-induced inhibition of C. parvum infection with an impairment in glutaminolysis, representing an important metabolic pathway in intestinal cells. The principal involvement of mTOR in C. parvum inhibition was confirmed by another mTOR-inhibitor (PP242, 0.5 µM), which also reduced C. parvum infection by 70–77%. Given that oleocanthal is not a selective mTOR inhibitor, we assume that this compound drives a multi-target-based inhibition of asexual C. parvum replication, amongst which mTOR is addressed.