Hemophilia A (HA) is associated with FVIII coagulation insufficiency or inactivity leading to excessive bleeding. Elevated FVIII, on the contrary, is associated with thrombophilia, thrombosis, myocardial infarctions, and stroke. Active FVIII (aFVIII) uses its C2 domain to bind to blood cells’ membranes, consequently carrying out its coagulative function. We developed a reliable flow cytometry (FC) method for FVIII detection that can be utilized for assessing surface-bound FVIII on leukocytes in different coagulation/clinical states; we analyzed 49 pediatric subjects, encompassing patients with HA, other coagulopathies, venous thrombosis, and normal coagulation. Interestingly, the total leukocyte surface FVIII showed a declining trend across thrombosis, normal, and hypo-coagulation states. As expected, the leukocytes of HA patients displayed significantly lower levels of cellular-surface FVIII in comparison to patients with thrombosis. However, no significant correlation was observed between circulating levels of FVIII in plasma and the levels of FVIII bound to leukocytes, indicating that the differences in FVIII surface binding are not directly proportional to the availability of FVIII in the circulation and suggesting a specific binding mechanism governing the interaction between FVIII and leukocytes. Intriguingly, when analyzing the distinct blood subpopulations, we observed that surface FVIII levels were significantly elevated in classical monocytes of thrombosis patients compared to HA patients, healthy controls, and patients with other coagulopathies. Our study highlights the reliability of our FC platform in assessing FVIII abundance on leukocytes’ membranes across coagulation states. Monocytes, particularly in cases of thrombosis, exhibit active binding of FVIII on their surface, suggesting a potential role in the pathophysiology of thrombosis that requires further investigation.

The immunological composition of the microenvironment has shown relevance for diagnosis, prognosis, and therapy in solid tumors but remains underexplored in acute leukemias. We investigated the significance of the acute myeloid leukemia (AML) bone marrow microenvironment in predicting chemosensitivity and long-term remission in pediatric patients. We analyzed 32 non-promyelocytic pediatric AML patients at diagnosis using a NanoString PanCancer IO 360 assay, RNA sequencing, and deep-phenotype flow cytometry analyses. The findings were validated using the pediatric TARGET AML dataset. A short signature of three interferon (IFN)-related genes (GBP1, PARP12, and TRAT1) distinguished patients with chemosensitive disease and reduced minimal residual disease after induction chemotherapy. The signature stratified patients overall, and within the clinically defined “standard-risk” group, patients with high gene expression at diagnosis had significantly longer overall survival. The leukemia microenvironment associated with this signature showed enrichment of non-exhausted CD4⁺ and CD8⁺ T cytotoxic lymphocytes and expansion of CD8⁺ T effector memory cells re-expressing CD45RA (TEMRA) in patients with a favorable prognosis. Our results show the importance of the bone marrow microenvironment in pediatric AML and provide tools for a refined stratification of “standard-risk” patients, lacking adequate risk-oriented therapies. They also offer a promising guide for tackling immune pathways and exploiting immune-targeted therapies.