Evidence for Ineffective Erythropoiesis in Patients with Severe Sickle Cell Anemia.

Peripheral destruction of sickled erythrocytes is recognized as the central pathologic mechanism of SCD. Less well established is the contribution of ineffective erythropoiesis to the pathophysiology of SCD. Patients with SCD frequently develop mixed hematopoietic chimerism after nonmyeloablative stem cell transplantation (NMA-SCT). This provides an opportunity to directly compare the differentiation and survival of normal donor and SCD erythropoiesis in vivo. To this end, total genomic DNA (gDNA) and erythroid-lineage specific chimerism in peripheral blood (PB) and marrow (BM) were analyzed in 5 patients (3 with SCD, 2 with non-SCD) following NMA-SCT with HLA-matched donors. RBC chimerism was measured by beta-globin RNA pyrosequencing, which directly and quantitatively sequences coding region polymorphisms in beta-globin RNA that distinguish recipient and donor-derived erythroid precursors. The clinical characteristics of the 5 patients are shown in the table below. All patients developed mixed gDNA chimerism, ranging from 18–50%, between days 60–180, with no differences between PB and BM levels. BM erythroid precursor chimerism, determined by Y chromosome FISH and ABO staining in 4 of 5 patients, were identical to total BM gDNA chimerism. PB from all 3 SCD patients had a ~2 fold greater level of donor-derived beta-globin RNA compared with total gDNA. These levels were similar for the 2 non-SCD patients. The presence of ineffective SS erythropoiesis was directly revealed by analysis of BM, which similarly revealed higher expression of donor-derived beta-globin RNA relative to the degree of donor erythroid progenitor engraftment in Pts 1–3. These findings were not present in Pts 4–5. Chimerism was determined in purified marrow populations of immature (pro-and basophilic –– glycophorin [GYPA+, CD71+ hi]) versus mature (polychromatophilic, orthochromatophilic [GYPA+, CD71+dim] ) erythroblasts to determine the stage of maturation at which SS erythroblasts are lost. While non-SCD patients demonstrated similar rates of donor and host erythropoiesis, all 3 SCD patients demonstrated progressive intramedullary loss of SS erythroblasts with maturation, with enrichment of donor erythroid precursor chimerism appearing from the earliest stages of hemoglobinization. The presence of ineffective erythropoiesis in SCD explains the maturation advantage of AA or SA donor erythroid precursor cells over SS cells that allows for greater donor contribution to overall erythropoiesis following SCT. This is the first definitive in vivo demonstration of ineffective erythropoiesis in SCD, and supports the notion that NMA-SCT can be curative for SCD in the setting of stable donor engraftment. Further studies examining the underlying mechanism of ineffective erythropoiesis in SCD are under investigation.

Patient clinical characteristics