71
OCTOBER 16-18, 2011 • PORTLAND, OR
P35
Improved Monochromatic Method for High Purity
Monocyte Gating by Flow Cytometry
Linda Wong, Bruce H Davis
Trillium Diagnostics, LLC, Bangor, ME, USA
Assays of antigen expression on myeloid cells have
an underlying premise that the assay integrates
high purity gating of the leukocyte subpopulation
in question. CD45/side scatter gating provides
suffcient gating purity for qualitative assays of antigen
expression; it is unsuitable for quantitative assays
of antigen changes, especially monocytes. We
have validated a monochromatic gating approach,
combining CD45 and CD64 labeled with the same
fuorochrome, that allows for high purity monocyte
gating. 25 blood samples were stained using three
different antibody combinations (CD45 FITC + CD163
PE; CD45 FITC + CD64 PE; CD45 FITC + CD64
FITC). Data analysis focused on the percentage of
“monocytes” defned by the various antibody and
side scatter gating combinations. Percent monocytes
recovered by monochromatic CD64 gating was
not statistically different from two color CD45 +
CD64 or CD45 + CD163 gating. All three methods
of immunologic monocyte identifcation yielded a
12 – 24% reduction in the monocyte percentage
compared to CD45/side scatter gating. Inter-observer
imprecision of monocyte percent decreased (CV of
> 7.7% to a CV of <4.3%)with monochromatic CD64
+ CD45 gating over standard CD45/side scatter
gating. A monochromatic combination of CD45 and
CD64 antibodies with scatter signals allows higher
purity monocyte gating by FC compared to CD45/
side scatter gating. This approach allows for the
development of a high resolution 4 color assay for
detection of PNH whereby a single 4 color tube will
allow simultaneous high purity monocyte (CD64+) and
neutrophil (CD15+) analysis of both PI linked protein
expression and FLAER binding.
POSTER ABSTRACTS