INTERMEDIATE MONOCYTE AND PLATELET ACTIVATION WITHIN THE EXTRACORPOREAL HEMODIALYSIS CIRCUIT AS NEW MARKERS FOR HEMODIALYSIS-ASSOCIATED THROMBOINFLAMMATION

During hemodialysis (HD), blood exposure to the extracorporeal circuit (ECC) triggers plasma protein and cell adhesion to the ECC and activates leukocytes, platelets, coagulation and complement pathways. Pro-thromboinflammatory signals spreading to the patient’s circulation contributes to systemic thromboinflammation, a major driver of cardiovascular (CV) disease, the leading cause of mortality in HD patients. Yet, the thromboinflammatory processes within the ECC—key drivers of systemic responses—remain poorly understood. This study aims to describe and quantify thromboinflammatory cell activation within the ECC.

In this exploratory pilot study, 3 clinically stable HD patients underwent 4-hours HD sessions via AV-fistula using heparin. Blood samples were collected pre- and post-HD. After blood retransfusion, the used ECC was rinsed with PBS-EDTA to retrieve circuit-adherent cells, followed by an enzymatic rinse (Accutase®) to detach rinsing-resistant cells. Cell counts (Alinity Hq, Abbott) and flow cytometry (FACSLyric, BD) were performed on both blood samples and rinse fluids. Scanning electron microscopy (SEM) of the used dialyzers was used to validate the efficacy of the rinsing. For each patient, SEM analysis of a used non-rinsed and EDTA-only rinsed dialyzer of a consecutive HD session was performed as control.  

3 patients (68 ± 13 years, 2 male) were included. The EDTA rinse yielded a median of 14.1 ×106 leukocytes [10.8–17.8 ×106] and 32.8 ×106 platelets [21.4–46.1 × 106]. Subsequent enzymatic rinse yielded a median of 15.3 ×106 leukocytes [5.29–21.1 × 106] and 45.5 ×106 platelets [44–62.4 ×106], despite being performed after blood retransfusion to the patient and EDTA rinsing. Flow cytometric analyses revealed a significant higher proportion of intermediate monocytes and activated platelets in rinse fluids compared to blood samples according to the CD14++CD16+ (intermediate monocyte subset), CD62P+ (platelet degranulation) and PAC1+ (platelet adhesion) expression respectively (Fig.1).  Neutrophils and monocytes were highly activated in both blood samples and rinse fluids, with CD66b and   CD11b expression reaching 100% in all conditions. SEM of a used non-rinsed fiber demonstrated feasibility of visualizing circuit-adherent cells. Remaining cell clusters persisted after EDTA rinsing, but not after enzymatic rinsing (Fig. 2).

Our findings demonstrate that rinsing of the ECC using an EDTA rinsing fluid only is insufficient to retrieve circuit-adherent cells. Our novel two-step protocol – EDTA followed by enzymatic rinsing - enables full retrieval, as visually confirmed by SEM. Further characterization demonstrates the sequestration of intermediate monocytes within the used ECC, a cell population associated to CV events and mortality in HD patients. Platelets, not explored as circuit-adherent cells before, were found to be abundant within the ECC and exhibited increased activation in the rinse fluids compared to blood samples. These findings supports that the ECC acts as a thromboinflammatory origin nidus. Given thromboinflammation’s role as a major driver of CV disease, our protocol offers a novel tool to investigate the cellular mechanisms underlying this phenomenon at ECC-level and enables future investigations into whether modifications to the HD setup can mitigate these responses.

This abstract was also submitted for the ERA25 congress and BVN annual meeting 2025.