Introduction: Sickle cell disease (SCD) is characterized by microvascular occlusion mediated in part by adhesion of sickle erythrocytes (SS RBCs) to the vasculature. Advanced flow adhesion (FA) technology facilitates SS RBC adhesion studies in conditions that simulate in vivo microvascular physiology. Most currently available FA systems measure SS RBC adhesion in non-pulsatile flow conditions, versus pulsatile blood flow conditions generated by the cardiac cycle. The influence of pulsatile blood flow on SS RBC adhesion may be particularly important in pediatric SS RBC adhesion, as children have a broad range of heart rates. This study compares SS RBC adhesion in non-pulsatile and pulsatile flow conditions, utilizing a commercially available, microfluidic FA system.

Methods: Peripheral blood was obtained from patients with homozygous SCD (n=7) in steady state (5–18 years) from the Sickle Cell Center at the Children's Hospital of Michigan. FA assays were performed in non-pulsatile and pulsatile flow conditions, at a shear stress of 1.0 dyne/cm², and increasing shear stress up to 20 dyne/cm²to assess avidity. A programmable control unit regulated pulse frequency, shear stress, and temperature. Adhesion was measured to immobilized human laminin and vascular cell adhesion molecule-1 (VCAM-1). A static adhesion assay was used to assess initrinsic adhesive properties of SS RBCs independent of flow dynamics.

Results: Standard assays were performed with 30 mL of isolated SS RBCs (1× 10⁷cells/mL), and SS RBC adhesion was comparable to levels previously reported in parallel plate flow adhesion assays. FA assays showed that adhesion to both laminin and VCAM-1 was significantly increased in the context of pulsatile blood flow (1.67Hz) compared to non-pulsatile blood flow by 8-fold and 56-fold, respectively. The relationship of SS RBC adhesion to increasing pulse frequencies was variable from patient to patient, although adhesion to both laminin and VCAM-1 was uniformly greater in all pulse frequencies tested (1.0, 1.5, 1.67, and 2.0 Hz) compared to non-pulsatile blood flow. When avidity of adhesion was tested 78% of SS RBCs remained adhered to VCAM-1 at the maximum 20dyne/cm² shear stress, whereas 6% of SS RBCs remained adhered to laminin at a shear stress of 20 dynes/cm². Pulsatile adhesion to VCAM-1 and laminin was unaffected by protein kinase A (PKA) inhibition, although adhesion to laminin decreased by 31% in one of three patients. To determine if increased adhesion under pulse-flow conditions was due to increased contact time with the immobilized substrate versus a change in the SS RBC's intrinsic adhesive state, we measured SS RBC adhesion in a static adhesion assay following exposure to pulsatile versus non-pulsatile conditions. There was no significant difference in static adhesion to VCAM-1, however adhesion of pulse-exposed SS RBCs to laminin was more variable. Static adhesion of pulse-exposed SS RBCs to laminin was reduced by 60% in the presence of a PKA inhibitor.

Conclusions: Our data demonstrate the application of a commercially available microfluidic flow adhesion assay system for efficient assessment of SS RBC adhesive properties. In the future, such advances may allow SS RBC adhesive properties to be evaluated clinically as a predictive tool for future vaso-occlusive events, and to predict individual patient response to anti-adhesive therapy. The small volume of blood required makes this system particularly attractive for studying pediatric samples. Additionally, our data demonstrate that adhesion to both an endothelial cell substrate (VCAM-1) and a subendothelial matrix substrate (laminin) is significantly influenced by the presence of pulsatile blood flow. Although PKA may play a minor role in pulsatile adhesion to laminin, increased contact time with immobilized laminin and VCAM-1 may be a greater contributor to increased adhesion under pulsatile conditions versus non-pulsatile conditions. Pediatric SS RBCs adhered to VCAM at higher levels and with more avidity compared to laminin. The pulsatile flow conditions described in this study more closely approximate in vivo microvascular conditions compared to non-pulsatile conditions commonly used to study SS RBC adhesion. Based on these differences in adhesion under pulsatile versus non-pulsatile flow, incorporating pulsatile flow in future adhesion studies may be more representative of in vivo conditions.

Disclosures: No relevant conflicts of interest to declare.