Acute ischemic stroke (AIS) remains a major cause of disability despite advances in reperfusion therapies (GBD 2021 Stroke Risk Factor Collaborators, 2021). Mechanical thrombectomy improves recanalization (Oliveira et al., 2022; Liu et al., 2020); however, patient outcomes vary widely, suggesting a role for secondary inflammatory mechanisms (Shen et al., 2023; Lattanzi et al., 2021; Aly et al., 2021). Myeloid cells, including neutrophils and monocytes, contribute to tissue injury, thrombosis, and reperfusion success, yet most studies focus exclusively on peripheral blood, overlooking immune processes occurring directly at the occlusion site and within the thrombus (Lin et al., 2025; Cao et al., 2024; Tsalta-Mladenov and Andonova, 2024; Otsu et al., 2023).

This gap limits understanding of local immunopathology and its relevance to clinical outcomes (Li et al., 2023; Liu et al., 2023; Li et al., 2025). The aim of this study is to compare immune profiles and transcriptomic signatures of myeloid cells across three compartments—arterial blood, peripheral blood, and thrombus—and to examine their associations with radiological markers and clinical recovery following thrombectomy.

This approach addresses a critical missing link between localized immune activation and patient prognosis, providing a framework for identifying biomarkers relevant to personalized stroke care.

This study will analyze three biological compartments:

Peripheral venous blood will be collected at two time points:

All samples will be immediately stabilized using appropriate preservation agents (e.g., RNAlater for nucleic acids, anticoagulants for cellular assays) and transported on ice to the laboratory. Sample processing will begin within 30 min of collection and include:

These procedures are designed to preserve the cellular integrity and molecular fidelity of all samples, ensuring reproducibility and reliability of downstream analyses. In addition, cytokine profiling will be conducted using the Luminex platform, and transcriptomic findings will be validated through quantitative real-time PCR (qPCR) and TaqMan Low-Density Arrays (TLDA) for broader gene expression analysis.

All clinical, radiological, and molecular data will be integrated to identify correlations between the patients' immunological profiles and treatment outcomes, as measured by the National Institutes of Health Stroke Scale (NIHSS), the modified Rankin Scale (mRS), and the Thrombolysis in Cerebral Infarction (TICI) score.

The study will be conducted between January 2026 and December 2029, based on the methodological framework developed during the 2024–2025 preparatory and pilot phase.

Figure 1 provides a schematic overview of the sample collection and processing workflow, detailing the timing, anatomical source, stabilization, and analytical steps for each biological compartment.

The study workflow, including sample collection, processing, and data integration, is summarized in Figure 1.

This multi-phase, multidisciplinary study leverages advanced immunological, molecular, and imaging technologies to comprehensively characterize the immune landscape of AIS in the setting of MT. By integrating clinical, radiological, histological, and multi-omics data—including a retrospective arm focused on archived thrombus tissue—the study aims to uncover robust biomarkers of stroke pathophysiology, therapeutic response, and clinical prognosis. The insights gained may guide the development of personalized treatment strategies and enhance outcomes in acute ischemic stroke care.

The preliminary results provide a solid methodological and biological foundation for the implementation of the project.

This statistical and methodological framework supports the implementation of the study: “Comprehensive Immunological Profiling in Acute Ischemic Stroke: Myeloid Cell Activation and Molecular Signatures in Blood and Thrombus during Mechanical Thrombectomy.” The combination of site-specific immune sampling, high-dimensional profiling, and rigorous data analysis is expected to yield novel insights into the immunopathology of acute ischemic stroke. The approach enables the identification of biomarkers predictive of stroke severity, therapeutic response, and long-term recovery, potentially guiding precision medicine strategies in stroke care.

Within this study, the only procedure exceeding routine clinical management is the collection of peripheral venous blood samples. This procedure involves minimal risk of minor adverse events (AEs), such as local pain, bruising, hematoma formation, minor bleeding, or local inflammatory reactions. These events are typically mild and self-limiting, requiring no additional interventions beyond standard care. All potential adverse events related to peripheral venous blood sampling will be systematically documented using a dedicated AE reporting form and classified according to severity (mild, moderate, severe), duration, and causality. Each AE will be categorized as unrelated, probably related, or related to the patient's participation in the research. Patients will be monitored during hospitalization and follow-up visits as part of standard clinical practice to identify potential delayed AEs (e.g., infection at the venipuncture site, delayed hematoma formation). Such events will also be included in the study documentation. In the unlikely event of a serious adverse event (SAE)—defined as any medical event resulting in death, hospitalization, permanent disability, or requiring significant medical intervention—the research team will immediately notify the Ethics Committee and consider withdrawing the patient from the study, prioritizing patient safety and welfare. Due to the observational nature and low-risk profile of this study, we deem the establishment of an independent Safety Review Board unnecessary. However, the principal investigators, supported by clinical neurologists and interventional radiologists, will critically review any ambiguous or severe AE cases to determine causality and manage patient safety effectively. The collection of arterial blood and thrombus material occurs routinely during standard mechanical thrombectomy procedures, and thus does not involve additional invasive interventions specific to this research. Consequently, any adverse events arising from these procedures would be attributable to standard therapeutic practice and not directly to research-specific activities.

All relevant clinical and research data—including clinical parameters, imaging studies, procedural outcomes (such as NIHSS, mRS, and TICI scores), and laboratory/immunological results—will be initially collected using standardized paper-based Case Report Forms (CRFs), which serve as the primary data source, and subsequently entered into a dedicated, secure electronic case report form (eCRF) system developed specifically for this study.

Clinical data routinely generated during patient care (e.g., imaging findings, procedural results, and clinical assessments) will be extracted from the hospital's electronic medical information system by trained and authorized study personnel. These data will be pseudonymized using unique subject identification codes before being recorded in the paper CRFs and later transferred into the eCRF.

Research-specific data generated through external laboratory analyses (e.g., immunological profiling, transcriptomics, microscopy) will be directly recorded in the eCRF by authorized personnel. All entries in the electronic system will be subject to audit trails to ensure traceability and data integrity.

The electronic database will be stored on encrypted, password-protected computers located on secure institutional servers, with access strictly limited to designated members of the research team, including data managers and statisticians. Regular data backups will be performed to prevent data loss, and access logs will be maintained to monitor any changes.

All physical (paper-based) documentation containing sensitive or identifiable patient information (e.g., signed consent forms, clinical reports) will be securely stored in a locked cabinet located in the office of Prof. Tadeusz Popiela. Access to this cabinet will be restricted to authorized personnel only.

Both physical and electronic records will be securely archived for a minimum of 5 years following the final publication of the study results, in accordance with institutional policies and applicable legal regulations, including the General Data Protection Regulation (GDPR).

Regular internal data audits will be conducted throughout the study to ensure data accuracy, completeness, and compliance with ethical, regulatory, and data protection standards.

To ensure consistency and high quality of collected data and analytical results, all study procedures—including blood and thrombus collection, sample processing, immunological assays, and molecular analyses—will be performed according to standardized operating procedures (SOPs) developed prior to study initiation. All personnel involved in sample acquisition and laboratory analyses will receive detailed practical training focused specifically on methodological aspects relevant to the study, such as sample stabilization, handling, labeling, transport, and advanced analytical techniques (spectral flow cytometry, RNA sequencing, microscopy techniques). Completion of training will be documented, and adherence to SOPs will be periodically verified. Prior to initiating the main phase of the study, pilot validation tests will be conducted using reference material to assess the accuracy, reproducibility, and analytical consistency of key laboratory procedures. To assess measurement consistency and reliability, the study protocol will include both intra-operator and inter-operator variability tests. Intra-operator reproducibility will be evaluated through repeated measurements performed by the same investigator on selected samples. Inter-operator reproducibility will be assessed by comparing results obtained independently by different investigators. Intraclass correlation coefficients (ICC) will be calculated separately for cytometric parameters (e.g., CD11b, CD66b expression), cytokine levels, RNA expression profiles, and cfDNA quantification. Throughout the duration of the study, the principal investigator will conduct regular review meetings with the research team to monitor study progress, address potential methodological issues, and verify data integrity. Additionally, periodic audits will be carried out by an independent Data Monitoring Committee to ensure compliance with ethical standards, study protocols, and good clinical practice (GCP) guidelines. All costs related to study-specific procedures, including laboratory analyses, sample processing, and additional tests required for the purposes of this research, will be covered by the research grant funding the project.

Angiographic reperfusion (TICI) is independently assessed by two interventional neuroradiologists blinded to immunological data. Functional outcomes at 90 days (mRS) are evaluated by certified neurologists not involved in the thrombectomy procedure, minimizing assessment bias.

This study has several inherent limitations and risks that should be considered when interpreting the results.

First, the single-center design and the intrinsic heterogeneity of thrombus composition limit the generalizability of the findings. Sampling variability, partial clot fragmentation during retrieval, and the limited availability of arterial blood in some cases may introduce bias. Moreover, arterial blood sampling at the site of occlusion was feasible only in patients with successful thrombus retrieval, potentially introducing selection bias toward individuals with more favorable anatomical or procedural characteristics. While the exclusion of IVT-treated patients may reduce generalizability, it enhances the interpretability of local immune signatures.

Technical risks include RNA degradation, hemolysis, and limited thrombus quantity. These risks are mitigated through strict collection protocols, immediate sample processing, and rigorous quality control.

Isolation-related risks, such as cell damage or ex vivo activation, are addressed by employing gentle dissociation procedures, completing cell isolation within 60 minutes, and validating markers of cell integrity and phenotype.

Biological variability, including etiological heterogeneity and confounding effects of medications (e.g., antiplatelet agents), represents an additional challenge. Stratification according to TOAST classification and the use of multivariable models help to minimize these confounding influences.

Animal model studies face risks such as variability in MCAO models and mortality in transgenic lines. These are mitigated through strict standardization of procedures and careful monitoring of cerebral blood flow.

Analytical risks include the integration of multidimensional data and the potential overinterpretation of observed correlations. These are addressed through mechanistic validation in animal models and rigorous statistical control.

Taken together, these limitations and risks highlight the complexity of translational thrombus research and underscore the need for careful interpretation of findings, replication in larger and multi-center cohorts, and complementary mechanistic studies.

This study represents a novel, translational investigation into the immune mechanisms underlying acute ischemic stroke. By combining direct sampling from the site of vascular occlusion with detailed immunological profiling and clinical correlation, the project aims to identify actionable immune biomarkers and pathways—particularly the MIF–RIPK1 axis—that influence stroke severity and response to treatment. The integrative design ensures scientific rigor, clinical relevance, and potential translational impact in the development of personalized stroke therapies.