Measurements of in vivo megakaryocytopoiesis: studies in nonhuman primates and patients.
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) IIb/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 microm (range: 21 microm for 2N to 56 microm for 64N cells), volume is 26 x 10(3) fL, and MK number is 10 x 10(6)/kg, giving a total MK mass of 26 x 10(10) fL/kg. The modal ploidy is 16N. In essential thrombocythemia patients (n = 10) with a mean platelet count of 907 +/- 23 x 10(6)/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 x 10(6)/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 microg/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 myeloid markers including CD36, CD45 and IgG-Fc gammaRII 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.
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Division of Hematology and Oncology, Emory University School of Medicine, Grady Memorial Hospital, Atlanta, Georgia 30303, USA.
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D000818:Animals; D001853:Bone Marrow / Q000502:physiology; D018955:CD36 Antigens / Q000096:biosynthesis; D002338:Carotenoids / Q000494:pharmacology; D002454:Cell Differentiation; D002455:Cell Division; D019070:Cell Lineage; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D019793:Fluorescein / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000096:biosynthesis; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D008253:Macaca mulatta; D008533:Megakaryocytes / Q000166:cytology* / Q000502:physiology*; D010215:Papio; D010799:Phycoerythrin / Q000494:pharmacology; D011003:Ploidies; D011994:Recombinant Proteins / Q000494:pharmacology; D013921:Thrombocytopenia / Q000097:blood / Q000188:drug therapy; D013997:Time Factors
D018955:CD36 Antigens; D006684:HLA-DR Antigens; D015850:Interleukin-6; D011994:Recombinant Proteins; D010799:Phycoerythrin; D003609:Dactinomycin; C016040:peridinin; D002338:Carotenoids; C025942:7-aminoactinomycin D; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017493:Leukocyte Common Antigens; D019793:Fluorescein