Patients With Paroxysmal Nocturnal Hemoglobinuria Bone Marrow Cd34 ~ + Cd59 On ~ + Cells And Of Cd34 ~ + ~ Cd59 On - Cells In Vitro Kuang Comparative Study Of Growth

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic anemia. The main manifestations are recurrent episodes of hemoglobinuria, chronic anemia and thrombosis. Recent studies have demonstrated that PNH is a clonal hematologic disorder characterized by the deficiency in cell surface expression of GPI-anchored proteins (GPI-AP) such as CD55 and CD59 which are responsible for the increased sensitivity of erythrocytes to complement, and the deficiency of GPI-AP is caused by somatic mutation in the PIG-A gene in hematopoietic stem cells. However, the fundamental nature of the disease that renders abnormal clones (GPI-AP~-) to be able to persistently coexist with normal clones (GPI-AP~+) in marrow and attain clonal dominance has not been fully elucidated. The present treatment for PNH is not very effective except for allogeneic bone marrow transplantation. We think we can separate normal stem and progenitor cells (CD34~+CD59~+) from abnormal clones (CD34~+CD59~-) in vitro and perform ex vivo expansion of CD34~+CD59~+ cells for hematopoietic reconstruction. The present study was undertaking ex vivo expansion of CD34~+CD59~+ cells, CD34~+CD59~- cells from patients with PNH and CD34~+ cells from normal control.CD34~+CD59~+ and CD34~+CD59~- cells were selected from the bone marrow mononuclear cells in patients with PNH by means of immunomagnetic cell sorting and flow cytometry cell sorting ( magnetic microbead-flow cytometry two step sorting method ) . The purities of CD34~+CD59~+ cells and CD34~+CD59~- cells were all above 95%. Ex vivo expansion of CD34~+CD59~+ cells showed that (1) FLT-3 ligand and thrombopoietin were identified that played important roles with regard to amplification of CD34~+CD59~+ cells. The basic combination of hematopoietic growth factors for ex vivo expansion was SCF+IL-3+IL-6+FL+TPO. (2) The best combination of...