Evidence for ineffective erythropoiesis in severe sickle cell disease

Abstract

Peripheral destruction of sickled erythrocytes is a cardinal feature of sickle cell disease (SCD). Less well established is the potential contribution of ineffective erythropoiesis to the pathophysiology of this hemoglobinopathy. Since patients with SCD frequently develop mixed hematopoietic chimerism after allogeneic nonmyeloablative stem cell transplantation, we used this opportunity to directly compare the differentiation and survival of SCD and donor-derived erythropoiesis in vivo. Donor and recipient erythropoiesis was compared in 4 patients with SCD and 4 without SCD who developed stable mixed hematopoietic chimerism following transplant. Molecular analysis of chimerism in peripheral blood and bone marrow demonstrated higher expression of donor-derived beta-globin RNA relative to the level of donor-derived genomic DNA in patients with SCD. Analysis of chimerism in immature (glycophorin A-positive [GYPA(+)], CD71(hi)) and mature (GYPA(+), CD71(neg)) erythroblasts confirmed the intramedullary loss of SS erythroblasts with progressive maturation. In patients with SCD, relative enrichment of donor erythroid precursors began to appear at the onset of hemoglobinization. Ineffective erythropoiesis of homozygous hemoglobin S (SS) progenitors thus provides a maturation advantage for homozygous hemoglobin A (AA) or heterozygous hemoglobin S/hemoglobin A (SA) donor erythroid precursor cells that results in greater donor contribution to overall erythropoiesis following stem-cell transplantation and improvement of clinical disease.

Figure 1.

Higher expression of donor-derived genomic DNA in peripheral blood from SCD patients. Peripheral blood genomic DNA and beta-globin RNA donor chimerism were compared between patients with SCD (A) and without SCD (B). ▪ indicates genomic DNA chimerism; , Beta-globin RNA chimerism.

Figure 2.

Recipient SS erythroblasts are selectively lost within the intramedullary space. Genomic DNA () and β-globin RNA (▪) donor chimerism derived from bone marrow mononuclear cells (BMMCs) were compared between patients with SCD (patients 1-4) or other diseases (patients 5-8) at day 100 following allogeneic stem cell transplantation.

Figure 3.

Early and late erythroblasts can be identified and isolated by flow cytometry. Immunophenotyping of marrow mononuclear cells with glycophorin A-PE and CD71-FITC can identify immature from mature erythroblasts, as confirmed by histochemical staining of the sorted populations (inset photographs). Samples were analyzed using an Olympus BX41 microscope equipped with a UPlanFL 40×/0.75 objective lens (Olympus, Melville, NY). Pictures were taken with Olympus QColor3, and were analyzed with QCapture 2.60 software (QImaging, Burnaby, BC, Canada) and Adobe Photoshop 6.0 software (Adobe, San Jose, CA).

Figure 4.

Comparison of donor chimerism. Comparison of donor chimerism between unsorted (), immature (▪), and mature () marrow erythroblasts isolated from marrow and peripheral circulating nucleated RBCs (□) between SCD patient 3 and non-SCD patient 5.