Current Clinical Practice: A New Approach to the Analysis of Apoptosis in the Leukemic Subpopulation by Flow Cytometry Using a CD45 Gating Strategy

Abstract

Bone marrow and peripheral blood are heterogeneous tissues containing cells of different hematopoietic lineages. It is possible to detect leukemic cells by flow cytometry using a gating strategy, which combines CD45 expression on the cell surface with right angle light scatter (SS). This approach was applied to 15 cases of AML. Myeloblasts had the lowest CD45 fluorescence intensity of any of the cells in the myeloid series and also had the lowest SS, approximately equivalent to monocytes, but greater than lymphoblasts and lymphocytes. Using this gating strategy in each sample we could identify up to 5 separate cell compartments. Our results showed good correlation between the flow differential and the manual differential cell count. However in some cases, especially when a sample became hypocellular, the flow differential was more sensitive in identifying leukemic blasts. Total apoptosis (i.e. apoptosis in all cell populations combined) varied during the treatment between 0–34%. In the blood, the highest percentage of total apoptotic cells usually occurred between day 3–5 of treatment. The percentage of apoptotic cells varied depending on the cell type on a percentage basis. The leukemic population was less likely to undergo apoptosis compared to the lymphocytes, monocytes and more mature myeloid cells. In normal cells, apoptosis occurred mostly in G₁ and S phases of the cell cycle. Apoptosis among CD45-blasts usually varied between 0–5%. Myeloblasts also had a tendency to undergo apoptosis in G₁ and S phases of the cell cycle. The CD45-blast apoptotic peak in the blood occurred between day 5–7 of treatment. Analysis of drug-induced apoptosis in bone marrow seems to provide more information than such measurements in peripheral blood.

Keywords:

• Acute myeloid leukemia
• drug-induced apoptosis
• cell cycle
• CD45