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50
ICCS 2012
P36
FLUOROCHROME CHOICE INFLUENCES ESCAPEE
FORMATION IN HIGH DIMENSIONAL FLOW CYTOMETRY
Richard D Schretzenmair, Andrew D Bantly, Lifeng Zhang,
Amy Steinmetz, Jonni S Moore
University of Pennsylvania
Introduction:
The apparent loss of mononuclear populations
during staining with fluorescent antibodies, the so-called
lymphocyte escapee phenomenon, was noted as early as
1994. The effect was most evident with FITC in ammonium
chloride lysed whole blood. Although there has been a
large expansion of fluorescent probes, it has not been
determined if the escapee phenomenon occurs with the
new fluorochromes and if it is restricted to certain subsets.
This information is critical to panel design and to accurate
measures of individual leukocyte subsets.
Materials
and Methods:
Pre-lysed whole blood was stained with
CD3 antibodies conjugated to more than 20 different
fluorochromes and analyzed on a BD FACSCanto with
11-color upgrade or a BC Gallios. Events were analyzed
using a pan-Leukocyte gate, excluding only debris and CD3
vs. SSC plots were generated. Escapees were classified
as events with CD3 intensity similar to T cells (CD3+ low
SSC) which had SSC properties more appropriate to that
of Granulocytes or Monocytes. In addition several other
pan-T cell, T cell subset and B cell markers were tested.
Results:
Escapee formation is evident with multiple probes
and is dependent upon the fluorochrome tag and not on
the antibody clone. Large difference between donors was
noted, some having virtually no escapees while others with
substantial numbers.
P37
IMMUNOPHENOTYPICAL FEATURES OF ACUTE
MYELOID LEUKEMIA CELLS IN ASSOCIATION WITH ITD
MUTATION IN GENE FLT3.
Konstantin Slobodnyuk,
Margarita Gorchakova, Margarita Ivanchenko, Irina Fridlin,
Mikhail Zaraiski, Yekaterina Zueva
State Pavlov Medical University
Background:
Immunophenotypical and genetic alterations
in acute myeloid leukemia have been demonstrated
to be important prognostic factors for the stratification
of the patients in the different risk groups. There are
some controversial data concerns on concordance
immunophenotypical pattern of leukemic cells in AML
patients with internal tandem duplication (ITD) in gene
FLT3. Objective: By using 5-colour ow cytometry and PCR
with detection in gel electrophoresis, we have studied the
association of immunophenotypical features of leukemic
cells in AML with the mutation FLT3-ITD.
Methods:
We have
studied leukemic cells of bone marrow from patients with
AML (n=45) and healthy controls (n=13). For surface labeling
antigens on blast cells we used monoclonal antibodies:
CD 2, 3, 4, 5, 7, 11b, 13, 14, 15, 16, 19, 20, 22, 33, 34, 36,
38, 41a, 56, 64, 71, 117, 184, HLA-DR (Beckman Coulter),
CD133 (Miltenyi Biotech). Cells were labeled according to
the standard procedures of manufactrory instructions. For
PCR amplification we used primers FLT3-ITD (forward):
5’-TCTGCAGAACTGCCTATTCCT-3’; FLT3-ITD (reverse):
5’-CTTTCAGCATTTTGACGGCAA-3’. For the gel of
electrophoresis we used 3% polyacrylamide.
Results:
Mutation FLT3-ITD was detected in 13 cases out of 45
(29%). We found signi cant difference (p<0,05) in the
number of leukemic blasts in the bone marrow that were
increased in the patients with the mutation FLT3-ITD at initial
diagnosis. Acute myeloid cells with the mutation FLT3-ITD
showed a signi cant increase in the surface expression of
antigens CD7 and CD13 respectively. Kaplan-Meier analysis
showed signi cant decrease in the overall survival in patients
POSTER ABSTRACTS