SEPARATION OF HUMAN MEGAKARYOCYTES BY STATE OF DIFFERENTIATION ON CONTINUOUS GRADIENTS OF PERCOLL - SIZE AND PLOIDY ANALYSIS OF CELLS IDENTIFIED BY MONOCLONAL-ANTIBODY TO GLYCOPROTEIN-IIB/IIIA

  • 1 May 1986
    • journal article
    • research article
    • Vol. 67  (5) , 1286-1292
Abstract
Human bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocytic lineage. Megakaryocytes were identified by indirect immunofluorescence using a monoclonal antibody (LJP4) specific to the glycoprotein IIb/IIIa (GPIIb/IIIa) complex of platelets. Neither endothelial cells nor monocytes expressed the epitope identified by this antibody. Simultaneous measurement of size and ploidy were made on 2,359 GPIIb/IIIa-positive cells. The smallest cells were located in the most dense fractions where 81% of all 2N and 66% of 4N cells were found at densities .gtoreq. 1.050 g/mL. The largest cells were detected in the least dense fractions, with 70% of 16N, 78% of 32N, and 100% of 64N cells found at densities < 1.050 g/mL. Ninety-four percent of all GPIIb/IIIa-positive cells < 14 .mu.m in diameter were found at densities > 1.050 g/mL. An exception to this inverse relationship was observed in the uppermost gradient fractions where an anomalous distribution of size and ploidy was found. Megakaryocytic viability was identified as being greater than 90% in all fractions. The data show that megakaryocytic differentiation as assessed by size and ploidy varies inversely with Percoll density. Separation of marrow on continuous Percoll gradients may be a useful method to separate megakaryocytes at different stages of differentiation.