Combining counterflow centrifugal elutriation and glycoprotein Ib-dependent purification of human megakaryocytes: efficacy and selectivity.
To estimate the efficacy of human megakaryocyte purification techniques, mixtures of known numbers of megakaryocytes with a known ploidy range and of bone marrow or peripheral blood mononuclear cells were made. These artificial bone marrow samples were submitted to either a counterflow centrifugal elutriation or Percoll density separation followed by the glycoprotein Ib-dependent agglutination procedure. Also crude bone marrow samples were submitted to counterflow centrifugal elutriation directly followed by a glycoprotein Ib-dependent agglutination. The counterflow centrifugal elutriation resulted in a mean megakaryocyte recovery of 81% (mean 81% +/- 2.3). Purification by glycoprotein Ib-dependent agglutination after either a Percoll density separation or counterflow centrifugal elutriation resulted in a recovery of 61% (mean 61% +/- 15%) and 81% (mean 81% +/- 6) respectively. Purity of the resulting material was 87% (mean 87% +/- 11) and 83% (mean 83% +/- 5) respectively. The various isolation procedures did not affect the ploidy distribution of megakaryocytes greater than or equal to 8N. Counterflow centrifugal elutriation was preferred as the preparing step before glycoprotein Ib-dependent agglutination because of the lower variability in recovery and purity of megakaryocyte populations. When large numbers of rather pure and mature megakaryocytes are required, counterflow centrifugal elutriation followed by the glycoprotein Ib-dependent agglutination is a relatively simple method to purify human megakaryocytes without an appreciable loss in ploidy class greater than or equal to 8N.
van Pampus
E C
EC
Free University Hospital, Department of Haematology, Amsterdam, The Netherlands.
van Geel
B J
BJ
Huijgens
P C
PC
Wijermans
P W
PW
Ossenkoppele
G J
GJ
Rodriguez
F
F
Zevenbergen
A
A
Langenhuijsen
M M
MM
D001854:Bone Marrow Cells; D002449:Cell Aggregation; D002469:Cell Separation / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000502:physiology; D010980:Platelet Membrane Glycoproteins
D010980:Platelet Membrane Glycoproteins