{"doi":"10.3389/fimmu.2025.1637038","title":"Experimental conditions shape in vitro formation of murine platelet-leukocyte aggregates","abstract":"Background: Platelets interact with leukocytes to fine-tune their functions, thus providing essential regulation of (patho-) physiologic immune responses in various diseases. Circulating platelet-leukocyte aggregates (PLAs) represent a sensitive biomarker to estimate disease severity both in patients and murine models. However, a limited understanding of the sensitivity of PLA measurements to methodological variables may undermine their accuracy and comparability. Objectives: To elucidate how blood draw techniques, anticoagulation, processing delay and assay temperature affect murine platelet-leukocyte interactions. Methods: or cardiac puncture, anticoagulated with heparin, citrate or acid-citrate-dextrose (ACD) +/- recalcification and stored for 30-120 min before stimulation at room temperature or 37°C with adenosine diphosphate (ADP), cross-linked collagen-related peptide (CRP-XL) and protease-activated receptor 4-activating peptide (PAR4-AP). PLA formation and leukocyte activation were analyzed by flow cytometry. Results: Basal PLAs were minimally affected by blood sampling and anticoagulant, though delayed processing significantly raised basal PLAs. Agonist-induced PLA formation was independent of anticoagulation, and sampling technique did not affect ADP- or PAR4-AP-induced PLA levels. However, CRP-XL sensitivity was elevated in blood obtained by cardiac puncture. Contrarily, both delayed processing and stimulation at 37°C impaired CRP-XL sensitivity, but augmented ADP and PAR4-AP responses. Regulation of leukocyte activation did not follow PLA patterns, with monocytes and neutrophils displaying distinct susceptibility to anticoagulation, storage and temperature. Conclusions: , underlining the critical need for fastidious assay optimization to support data reproducibility and comparability.","journal":"Frontiers in Immunology","year":2025,"id":536618,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9625,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":669044,"name":"Alice Assinger","orcid":"0000-0002-5670-5910","position":1,"is_corresponding":false},{"id":711340,"name":"Waltraud C. Schrottmaier","orcid":"0000-0003-0550-4120","position":2,"is_corresponding":false},{"id":1391493,"name":"Silvia Maria Grazia Trivigno","orcid":null,"position":0,"is_corresponding":true}],"reference_count":66,"raw_metadata":null,"created_at":"2026-07-19T02:52:05.227140Z","pmid":"40963605","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}