Erythroid-Specific Chimerism Analysis after Allogeneic-Hematopoietic Stem Cell Transplantation Using a Human Leukocyte Antigen-Identical Sibling Donor in Sickle Cell Disease Patients

Introduction: Allogeneic-hematopoietic stem cell transplantation (HSCT) using a human leukocyte antigen (HLA)-identical sibling donor is a proven cure for Sickle Cell disease (SCD). At least 20% donor myeloid chimerism in peripheral blood (PB) may be sufficient to reverse the sickle phenotype after HSCT. Here, we sought to implement an erythroid-specific chimerism analysis in order to decipher kinetics of lineage-specific engraftments and correlated biological and clinical phenotypes.

Method: Adult SCD patients who underwent an allogeneic non-myeloablative HSCT from HLA-matched related donors were included between 2020-2024 in our referral center Henri Mondor Hospital. Data were analyzed at 6 months post HSCT. Erythroid-specific chimerism was assessed in PB by flow cytometry using specific anti-Human-b^S-globin and anti-Human-b-globin fluorescent monoclonal antibodies. Erythroid-specific chimerism was assessed in mature CD71-negative red blood cells (RBC) and in CD71+ reticulocytes. In parallel, donor chimerism was assessed in total white blood cells (WBC) and sorted CD3+ lymphocytes. Hemolytic follow-up included Hb level, reticulocytes count, plasma lactate dehydrogenase (LDH); an extended panel was proposed with measures of free plasma-Hb, heme and hemopexin by spectrophotometric assays. Event-free survival (EFS) at 6 months was defined as the probability of being alive with sustained donor cell engraftment (HbS<30%) and no vaso-occlusive event (VOE).

Assuming that donor CD 71+ is correlated with level of erythroid engraftment, patients with a sustained donor engraftment (HbS<30%) was classified as total (>80% donor CD71+ chimerism) or partial engraftment (20-80% donor CD71+ chimerism). Graft failure (GF), graft-versus-host disease (GVHD), [HbS] and overall survival (OS) were also analyzed.

Results: Eleven patients were analyzed at 6 months post-HSCT. Homozygous SS genotype was showed in 82%; median age at transplantation was 35 years-old [28-42]. Patients had been previously treated with Hydroxycarbamide (92%) and/or chronic transfusion therapy (42%). Indications of HSCT were repetitive VOE in 83% and/or intra-cranial cerebral vasculopathy in 25% of patients. GF occurred in one S-beta₀ Thal patient at 12 months (donor chimerism WBC at 17%; HbS at 35%) and VOEs reoccurrence. In this case, GF could be predicted by donor CD71+ chimerism at 1-month post-HSCT (20%) but not with donor chimerism measured at the same date in WBC (69%) and CD3 + (40%).

Median period of aplasia was 7.5 days [5.7-13.5] based on neutrophil count (>0.5×10⁹/L); platelet count was never observed below 20x10⁹/L. No VOE was observed except for the GF patient. Probabilities of EFS and OS at 6 months post-HSCT were 92% and 100%. One acute GVHD stade II was observed.

At 6 months post HSCT, median RBC and reticulocyte CD71+ donor chimerism was 99% [97-100] (n=12) and 98% [42-100] (n=12) respectively; median donor chimerism in total WBC was 87% [50-96] (n=12) and 23% [8-50] in CD3+ cells (n=10). Median HbS was 14.6% [0.8-36.3]. Hb was 11.8 g/dL [9.9-12.2] at 6 months of follow-up; reticulocyte count was 42x10⁹/L [36-61]; LDH was 342 U/L [274-479]. Extended hemolysis panel at last follow-up 24.8 months [6.0-34.1] showed plasma free-Hb, heme and hemopexin median concentration at 4,4 µM (3.4-11), 0.15 µM (0.1-0.24) and 4.3 µM (1.4-8.8). Hemopexin was lower in partial engraftment group (p<0.05); no differences were observed between total and partial engraftment groups as regards to plasma free-Hb and heme concentrations (p>0.05).

Among HSCT with sustained donor cell engraftment at 6 months (n=11), total engraftment (donor CD71+ >80%) was observed in 9/11 (82%) with median donor chimerism in CD71+ at 99% [94-100], 100% [99-100] in RBC and 94% [84-97] in WBC. Two patients were considered as “partial” engraftment (donor CD71+ chimerism between 20-80%) with median donor chimerism of 28% in CD71+ (n=2), 95% in RBC (n=2) and 41% in WBC (n=2). Donor chimerism measured in RBCs was >95% in all patients included partial engraftment. Donor WBC chimerism was significantly correlated with donor CD71+ chimerism (r= 0.77; p<0.001).

Conclusion: Erythroid specific donor chimerism measured in immature CD71-positive reticulocytes could be a good predictor of engraftment in SCD patients who underwent an allogeneic non-myeloablative HSCT. Hemolytic-long-term follow-up is required especially in patients with partial engraftment.