metrizamide and Myelodysplastic-Syndromes

Knowledge of the blast phenotype in myelodysplastic syndrome (MDS) would be valuable, as in other malignancies, but remains sparse. This is mainly because MDS blasts are a minor population in clinical samples, making analysis difficult. Thus, for this blast phenotype study, we prepared blast-rich specimens (using a new density centrifugation reagent for harvesting blasts) from blood and marrow samples of 95 patients with various MDS subtypes and 21 patients with acute leukemia transformed from MDS (AL-MDS). Flow cytometry revealed that a high proportion of the enriched blast cells (EBCs) from almost all patients showed an immunophenotype of committed myeloid precursors (CD34(+)CD38(+)HLA-DR(+)CD13(+)CD33(+)), regardless of the disease subtype. The cytochemical reaction for myeloperoxidase was negative in 58% of the cases. Thus, the EBC phenotype is more immature in MDS than in de novo acute myeloid leukemia. MDS EBCs often coexpressed stem cell antigens and late-stage myeloid antigens asynchronously, but rarely expressed T- and B-lymphoid cell-specific antigens. Markers for myeloid cell maturation (CD10 and CD15) were more prevalent on EBCs from low-risk MDS (refractory anemia [RA] and RA with ringed sideroblasts), whereas markers for myeloid cell immaturity (CD7 and CD117) were more prevalent on EBCs from high-risk MDS (chronic myelomonocytic leukemia, RA with excess blasts [RAEB], and RAEB in transformation) and AL-MDS. A shift to a more immature phenotype of EBCs, accompanying disease progression, was also documented by sequential phenotyping of the same patients. Further, CD7 positivity of EBCs was an independent variable for a poor prognosis in MDS. These data represent new, valuable information regarding MDS.

Topics: Acute Disease; Aged; Antigens, CD; Case-Control Studies; Cell Separation; Centrifugation, Density Gradient; Disease Progression; Female; Flow Cytometry; Humans; Immunophenotyping; Leukemia; Male; Metrizamide; Middle Aged; Myelodysplastic Syndromes; Phenotype; Prognosis; Survival Analysis

Myeloblast-rich samples, required for investigation of myeloid malignancies, can be obtained only during the untreated stage of leukemia. Existing methods for myeloblast enrichment have various prerequisites that limit their application. In this new method, a mixture of peripheral blood (Mixed PB) from an acute myeloid leukemia (AML) patient and from a healthy control containing 5% myeloblasts was subjected to density gradient centrifugation using a 14.5% metrizamide solution. Both high purity (86.3% +/- 1.5%) and high recovery of viable myeloblasts were achieved. Close to 100-fold blast enrichment, even from Mixed PB containing only 0.15% myeloblasts, was achieved. Similarly, this method highly enriched myeloblasts from unprocessed samples, including marrow cells, from patients with AML, myelodysplastic syndromes, and chronic myeloid leukemia (purity: 2.7% +/- 2.0% before separation, 56.6% +/- 28.3% after separation) (n = 22). The enriched blasts were suitable for various analyses, eg, flow cytometry, immunocytochemistry, cytochemistry, fluorescence in situ hybridization, and gene analysis.

Topics: Acute Disease; Bone Marrow Cells; Cell Separation; Centrifugation, Density Gradient; Flow Cytometry; Histocytochemistry; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Metrizamide; Myelodysplastic Syndromes; Myeloid Cells