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Abstract Introduction:Sickle cell disease (SCD) is caused by a mutation in the β-globin gene (HBB) that causes red blood cells (RBC) to sickle, adhere to the endothelium, and hemolyze prematurely. Even with current FDA-approved gene therapies, allogeneic hematopoietic cell transplantation (HCT) remains the most accessible curative option. The increasing use of haploidentical donors has expanded transplant access, especially among populations historically underrepresented in donor registries. Engraftment is associated with resolution of SCD-related symptoms, including the absence of pain episodes, normalization of hemoglobin levels, and reversal of hemolysis. The effect of HCT on RBC functional biomarkers remains poorly understood, with current monitoring primarily focused on neutrophil and platelet recovery. Further, it is important to identify early predictors of long-term clinical outcomes that can be applied to HCT. We assessed flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA-WB-VCAM) and the dynamic sickling assay (DSA) to offer insight into HCT efficacy. Methods: Whole blood citrate samples were collected from 8 SCD patients at baseline (BSL, pre-HCT), 3 months, 6 months, and 1 year (3M,6M,1Y) post-HCT, and 8 matched donors (BSL) enrolled in NHLBI HCT protocol 17-H-0069 or 000539-H. FA-WB-VCAM utilizes a microfluidic well-plate system with channels functionalized with VCAM; data are reported as cells/mm2. DSA samples are exposed to a proprietary enzymatically-induced hypoxic environment, time-lapsed imaging, and AI-based SICKLE™ image analysis software evaluating RBC sickling. Parameters measured include max sickling (%), AUC10 (%*min), and rate of sickling (%/min). All values presented as mean ± deviation; paired t-tests were performed, p<0.05 considered statistically significant. Results: Preliminary analysis reports 4 patients with successful engraftment and 4 with graft failure (GF) following HCT. In successfully engrafted patients, FA-WB-VCAM is significantly decreased from BL to 3M (291 ± 48 to 53 ± 51 cells/mm², p=0.02, n=4), and BL to 1Y (64 ± 46 cells/mm2, p=0.01, n=4). There was a significant decrease from 6M to 1Y although not 3M-6M. Interestingly, GF patients show a decreasing trend from BL to 3M (332 ± 243 to 122 ± 137 cells/mm2, p=0.26, n=4) and at 1Y (112 ± 0 cells/mm2, n=1) but not at 6M (392 ± 304 cells/mm2, p=0.94, n=3). Also, successful engraftment significantly improved sickling kinetics, evidenced by a statistically significant reduction in AUC₁₀ values at 3- (from 319 ± 87 %*min to 24 ± 15 %*min, p=0.004, n=4), 6- (35 ± 46 %min, p=0.006, n=4), and 12-months (41 ± 41 %min, p=0.008, n=4) post-engraftment. In GF patients, AUC10 showed a decreasing trend from BL to 3M (272 ± 75 to 26 ± 16 %min, p=0.01, n=4). Worsening of sickling kinetics was observed at 6M (157 ± 138 %min, p=0.48, n=3). Only one datapoint at 1Y was available for analysis (64 %min; n=1). Similar trends were also noted for max sickling and the rate of sickling. In patients with successful engraftment, we observed normalization of RBC function: decreased adhesion and reduced sickling three months post-HCT. Graft failure (GF) patients exhibited increases in adhesion (FA-WB-VCAM) and sickling kinetics at six months. Conclusions: RBC health normalizes following engraftment as evidenced by statistically significant reductions in adhesive properties and sickling kinetics as early as 3 months. GF patients do not maintain reduced levels of adhesion and RBC sickling parameters after 3M. A larger sample size and a longer post-HCT follow-up period is needed to establish whether normalization of RBC function is maintained. These findings suggest that FA-WB-VCAM, and DSA could serve as early indicators of RBC health normalization, engraftment success and assessing long-term efficacy of gene-therapy strategies. Followup studies are underway to determine the ability of these biomarkers to predict long-term clinical outcomes.