Ineffective Hematopoiesis in Megaloblastic Anemia Is Associated with Higher Frequency of Apoptosis in Each Lineage Immature and Mature Cells.

Oyake, Tatsuo; Ito, Shigeki; Kowata, Shugo; Murai, Kazunori; Ishida, Yoji

doi:10.1182/blood.V106.11.3601.3601

Abstract

A deficiency of Vitamin B12 is a major cause of megaloblastic anemia (MBA). Ineffective hematopoiesis is observed in MBA, characterized by cytopenia, bone marrow cells with dysplastic change and normal to hypercellularity. We reported that excessive apoptosis of each lineage CD34(+) cells was observed in myelodysplastic syndrome (MDS) in the last ASH meeting. In this study, we investigated the hypothesis that excessive apoptosis induced the ineffective hematopoiesis in MBA. We performed the three color flow cytometric analysis of bone marrow mononuclear cells in 12 MBA patients using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-each lineage antibody (anti-glycophorin A (GPA) antibody, anti-CD33 antibody and anti-CD41 antibody). The frequency of apoptosis in subpopulations of immature (CD34(+)) and each lineage (+) cells or those of mature (CD34(−)) and each lineage (+) cells were calculated as the ratio (%) of (cell number with Annexin V(+)) divided by (cell number in the subpopulation). The subpopulations include CD34(+)GPA(+) (immature erythroid), CD34(+)CD33(+) (immature myeloid), CD34(+)CD41(+) (immature megakaryocytic), CD34(−)GPA(+) (mature erythroid), CD34(−)CD33(+) (mature myeloid) and CD34(−)CD41(+) (mature megakaryocytic) cells. Much higher frequency of apoptosis was observed in each lineage CD34(+) cells in MBA (median: 23.8% (range: 10.8–43.6%) in erythroid, 43.5% (12.7–67.3%) in myeloid, 50.1% (21.0–64.1%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineages, respectively, n=10). While, the relatively higher frequency of apoptosis was observed in each lineage CD34(−) cells in MBA patients (median: 15.9% (range: 5.1–20.6%) in erythroid, 16.4% (5.6–23.2%) in myeloid, 16.1% (10.2–24.8%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (4.8% (1.3–6.6%) in erythroid, 2.2% (0.6–4.4%) in myeloid, 3.3% (1.5–7.1%) in megakaryocytic lineages, respectively, n=10).

These results suggest that the excessive apoptosis occurs not only in CD34(+) but also in CD34(−) cells, which induces ineffective hematopoiesis in MBA.

Figure 1.