Assessment of normal erythropoiesis by flow cytometry: important considerations for specimen preparation

Abstract

The extension of quantitative flow cytometric studies to the erythroid lineage in patients with suspected myelodysplastic syndrome has prompted a reassessment of cell surface antigen expression during normal erythropoiesis. Erythropoiesis in normal and pathologic bone marrows was studied to determine the expected antigenic relationships of maturing erythroid cells. A total of 200 bone marrow specimens were evaluated by multidimensional flow cytometry (MDF). Samples were prepared using either NH4 Cl lysis or Ficoll density gradient separation. Normal erythroid development is described as a two-step process observable with the intensity relationships between CD235a, CD71, CD45, CD105, CD34, CD117, and CD36. The variability of these intensities (CV) was determined. A comparison of processing techniques determined lysis is the optimal analytic technique for the analysis of early-stage erythroid cells. Nucleic acid staining with DRAQ5 revealed that Ficoll allows for the analysis of reticulocytes and mature erythrocytes otherwise eliminated by lysis. These data demonstrate while lysis alters the light scatter characteristics of erythroid precursors, it did not alter quantitative antigen expression or nucleic acid content. The expected variability in antigen intensities is defined. These studies provide a basis for a comparison of erythroid development between normal individuals and those with erythroid dysplasia associated with myelodysplastic syndromes.