59
OCTOBER 16-18, 2011 • PORTLAND, OR
P23
Improvement In Accuracy Of Clinical Lymphocyte
Subset Quantitation By Flow Cytometry Using A
7-Color Assay Incorporating CD14
Stacy C League, Xiangyang Dong, Prabin Thapa,
Roshini S Abraham
Mayo Clinic, Rochester, MN, USA
Introduction:
Flow cytometry is considered the
gold standard method for detailed lymphocyte
subset quantitation (T, B and NK cells). Previously,
we observed that the absolute CD45 lymphocyte
counts were consistently greater than the lymphosum
of individual T, B, and NK cell populations due to
the unintentional inclusion of monocytes in the
lymphocyte gate (Block et al 2010). Additionally,
over a 3-year time period, the median difference
of observed CD3, CD4, and CD8 counts (T-Sum)
went from being less than the calculated value to
approaching zero, indicating improved concordance
between the observed and calculated results and
the interquartile ranges also decreased suggesting
reduced analytical variability over time due to
operator training and experience. An alternative assay
was developed which included CD14, optimized
fuorochrome choices, and simplifed gating strategies
with the goal of improving the accuracy of clinical fow
cytometric lymphocyte subset quantitation.
Materials
and Methods
: Flow cytometry analysis for TBNK
quantitation was performed on 213 samples using
2 different fow cytometers and analytical software:
Current assay (BD 6-color Multitest®, BD FACS
Canto instruments with Canto software v2.4) and the
newly developed assay (7-color, Gallios instrument,
Beckman Coulter, Kaluza v1.2 software).
Results:
Linear regression analysis was performed for
absolute CD45, CD3, CD4, CD8, CD4+CD8+, CD19+,
CD16+56+, lymphosum, and T-Sum with the following
results: 0.96, 0.96, 0.96, 0.57, 0.65, 0.97, 0.96, 0.55,
and 0.67 respectively. Additionally, paired t-test
analysis revealed signifcant instrument and software-
related differences with a p <0.0001 for each of
these values.
Conclusions:
Inclusion of CD14 as an
exclusionary marker, use of optimized fuorochromes,
choice of fow cytometry instrumentation and
simplifed gating strategies signifcantly increased the
accuracy of clinical lymphocyte subset quantitation.
POSTER ABSTRACTS