Pilot Study to Establish a Repertoire of Megakaryocyte Markers, Quantifiable by Spectral Flow Cytometry, in Bone Marrow and Circulating in Patients Undergoing Cardiac Surgery for Bypass Grafting

Pilot Study to Establish a Repertoire of Megakaryocyte Markers, Quantifiable by Spectral Flow Cytometry, in Bone Marrow and Circulating in Patients Undergoing Cardiac Surgery for Bypass Grafting

Megakaryopoiesis, the mechanism of blood platelet production, is difficult to assess in clinical practice as it relies solely on microscopic description. Recent studies described the presence of emergency megakaryopoiesis bypassing the classical maturation stages under certain conditions. Circulating megakaryocytes were identified in inflammatory and hemostatic conditions. The aim of this study is to characterize the phenotype of medullar and circulating megakaryocytes by flow cytometry during and after coronary artery bypass grafting.

Blood platelets, known for their role in primary hemostasis, are produced through the fragmentation of bone marrow megakaryocytes (MKs). In recent years, the characterization of new platelet functions, particularly in innate immunity and inflammatory processes, has reshaped the paradigm of sequential bone marrow megakaryopoiesis. Indeed, numerous studies provide evidence of a more direct, so-called "emergency" platelet production, which bypasses the successive stages of megakaryocyte differentiation. Furthermore, megakaryopoiesis may not be exclusively marrow-based; splenic and pulmonary sites are also described, along with circulating megakaryocytes in peripheral blood. These circulating megakaryocytes are gaining interest due to their easier accessibility and specific characteristics, notably pro-inflammatory properties.

However, to date, clinical practice analysis of megakaryocytes remains limited to simple microscopic description. Given recent observations as outlined above, a more precise and in-depth description of megakaryocytes, whether marrow-based or blood-based, appears essential for understanding pathophysiological mechanisms. Flow cytometry (FC) coupled with spectral technology overcomes the traditional limitations of FC. This technique has allowed our team to describe different stages of marrow megakaryocyte maturation using a murine model of platelet pathology.

In this context, cardiac surgery with extracorporeal circulation for coronary artery bypass grafting could serve as a model for inflammatory and hemostatic challenges. Post-procedural thrombocytopenia is common and is partly correlated with the duration of extracorporeal circulation (ECC), which could be associated with thrombo-inflammation responsible for acute kidney injury or stroke. Due to both the physiological bone marrow challenge posed by this procedure and the ease of bone marrow collection inherent to this technique, this situation seems well-suited for conducting this feasibility study.

Currently, no study has described the impact of megakaryocyte phenotype on platelet changes induced by cardiac surgery, whether these changes are purely quantitative or associated with a deleterious pro-inflammatory post-procedural context.

Based on these elements, combined with the expertise we have gained from studying the megakaryocyte/platelet axis in small animals using spectral flow cytometry, we propose:

• To validate the established antibody panel that allows the identification and quantification of the different maturation stages of bone marrow megakaryocytes,
• To apply this panel to circulating megakaryocytes before and after the surgical procedure,
• To correlate the observations with the potential inflammatory signature for each patient.

The rationale for this pilot study is to consider circulating megakaryocytes as a new, accessible, and highly specific indicator of thrombo-inflammatory processes.

Inclusion Criteria:

- Age over 18

• Indication for programmed cardiac surgery, specifically coronary artery bypass grafting with sternotomy
• Up to date social security coverage
• Patients able to understand the purpose and constraints of the research project

Exclusion Criteria:

• Refusal to participate in the study,

• Pregnant and/or breastfeeding women (clinical interview),

• Unscheduled cardiac surgery, Acquired hematologic disorders (acute leukemia, lymphomas, myelodysplastic syndromes, and myeloproliferative syndromes)

  • Treatment with thrombopoietin receptor agonists
  • Treatment with anti-inflammatory (included corticoids) or/and immunosuppressors
  • Hematopoietic stem cell transplantation or organ transplantation
  • Active cancer with or without treatment (in remission, ongoing or treated within the last 6 months),
  • Palliative care patients
  • Any other type of cardiac surgery and previous sternotomy,
  • History of external thoracic radiotherapy,
  • Patient under legal protection (curatorship, guardianship or safeguard of justice)
  • Patients with ongoing clinical trial requiring collection of additional blood or bone marrow samples