Measurements of in vivo megakaryocytopoiesis: Studies in nonhuman primates and patients

Aaron Tomer, Laurence A. Harker

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

In vivo megakaryocytopoiesis was directly analyzed for megakaryocyte (MK) number and mass, expression of lineage-specific and myeloid differentiation markers, and cell maturation as determined by size, granularity and ploidy. Using a rapid method for multiparameter correlative analysis with three-color flow cytometry (FCM) and a single-argon-ion-laser analyzer, cell DNA in aspirated marrow was stained with 7-amino-actinomycin D, and surface membrane receptors were analyzed with antibodies and cytokines labeled with fluorescein, phycoerythrin and peridinin chlorophyll protein. MKs expressing glycoprotein (GP) Hb/IIIa were enumerated in relation to the nucleated erythroid precursors expressing glycophorin A, and MK diameters were measured by time-of-flight technique. In human marrow (n = 10) the average MK diameter is 37 μm (range: 21 μm for 2N to 56 μm for 64N cells), volume is 26 × 103 fL, and MK number is 10 × 106/kg, giving a total MK mass of 26 × 1010 fL/kg. The modal ploidy is 16N. In essential thrombocythemia patients (n = 10) with a mean platelet count of 907 ± 23 × 106\L, MK number and volume increased twofold with modal ploidy of 32N, and MK mass fourfold the normal value. After reducing the platelet count to 353 ± 42 × 106\L with anagrelide therapy, MK number and volume decreased with modal ploidy of 16N, resulting in reduced MK mass by 50%. By contrast, patients with chronic myelogenous leukemia (n = 3) showed an increase in small MKs with a modal ploidy of 8N. In non-human primates, treatment with interleukin 6 or GM-CSF increased MK volume and ploidy with a variable increase in cell number and platelet counts. Treatment with recombinant human MK growth and development factor (n = 6, 5 μg/kg for 28 days) increased platelet count fivefold, MK number fourfold, MK volume twofold and total mass sevenfold. Using three-color FCM, marrow MKs labeled for GPIIb/IIIa and stained for DNA expressed high levels of von Willebrand factor with a high resolution of 2N/4N MKs from the total marrow cells. The expression of mycloid markers including CD36, CD45 and IgG-FcγRII CDw32 correlated directly with increasing cell maturation, concordant with the expression of GPIIb/IIIa and GPIb. Conversely, the expression of HLA-DR declined with maturation. We conclude that pathophysiologic and therapeutic changes in megakaryocytopoiesis in vivo are readily quantified using FCM measurements.

Original languageEnglish
Pages (from-to)18-30
Number of pages13
JournalStem Cells
Volume14
Issue numberSUPPL. 1
DOIs
StatePublished - 1 Jan 1996
Externally publishedYes

Keywords

  • Flow cytometry
  • Megakaryocytopoiesis
  • Ploidy
  • Primary thrombocytosis
  • Thrombocytopenia
  • Thrombocytopoiesis
  • Thrombopoietin

ASJC Scopus subject areas

  • Molecular Medicine
  • Developmental Biology
  • Cell Biology

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