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

Purpose: Blasts isolated from bone marrow aspirates or blood samples of pat ients 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 sa mples were also analyzed. Methods: Blasts were isolated by standard density centrifugation. RBC were prepared from the pellet of the same centrifugati on. MTX-Glu(n) were analyzed by means of HPLC and radiochemical quantificat ion. Results: In lymphoblasts isolated from blood, the distribution pattern s 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 sam e patient. RBC accumulated MTX but no formation of MTX-Glu(n) occurred. Con clusions: The determination of MTX and MTX-Glu(n) in lymphoblasts isolated from blood samples of patients with common ALL provides qualitative informa tion on the capacity of the blasts to form MTX-Glu(n) since distribution pa tterns 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 lymph oblasts. Blood lymphoblasts are therefore not useful for a quantitative ana lysis 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 ma rrow can therefore be ignored.