Comparative analysis of methotrexate polyglutamates in lymphoblast preparations from bone marrow and blood, and the contribution of residual red blood cells

Abstract

Purpose: Blasts isolated from bone marrow aspirates or blood samples of patients with acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) were compared for uptake of methotrexate (MTX) and formation of MTX polyglutamates (MTX-Glu _n ). Red blood cells (RBC) from the same patient samples were also analyzed. Methods: Blasts were isolated by standard density centrifugation. RBC were prepared from the pellet of the same centrifugation. MTX-Glu _n were analyzed by means of HPLC and radiochemical quantification. Results: In lymphoblasts isolated from blood, the distribution patterns of MTX-Glu _n were the same as in bone marrow lymphoblasts, but the total amount of MTX-Glu _n accumulated in blood lymphoblasts was reduced by 41%–51% when compared to the same number of bone marrow lymphoblasts of the same patient. RBC accumulated MTX but no formation of MTX-Glu _n occurred. Conclusions: The determination of MTX and MTX-Glu _n in lymphoblasts isolated from blood samples of patients with common ALL provides qualitative information on the capacity of the blasts to form MTX-Glu _n since distribution patterns of MTX and MTX-Glu _n parallel that of bone marrow lymphoblasts. The amounts of MTX-Glu _n accumulated, however, were much lower in blood lymphoblasts. Blood lymphoblasts are therefore not useful for a quantitative analysis of MTX-Glu _n . The contribution of RBC to MTX and MTX-Glu _n in vitro is only marginal and residual RBC in lymphoblast preparations from bone marrow can therefore be ignored.