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component (analysis and interpretation) performed at
separate locations. Our site acquires samples on a
GalliosTM and the data is analyzed off site using InfinicytTM.
Compensation samples acquired on the GalliosTM using
AutoSetup settings did not appear to look optimally
compensated. We manually set the compensation settings
by aligning the MFI of the dual negative to the positive PE
and PC-5 positive populations, respectively. Both sets of
data were sent to our professional site for analysis. It came
to our attention that the results of the sample acquired on
the GalliosTM that appeared to have acceptable fluorescent
compensation did not look acceptable by the flow techs
analyzing the data with InfinicytTM. Our investigation
found that data acquired with the GalliosTM Baseline
Offset function “ON” displayed differently in CXPTM than in
InfinicytTM software. We also discovered that the InfinicytTM
Negative Visibility feature changes the display of data post-
acquisition. Without using post-acquisition compensation,
FCS Express displayed data similarly to CXPTM (it does
recognize the Baseline Offset function) while Beckman
Coulter’s KaluzaTM Software displayed data similarly to
InfinicytTM (it does not recognize the GalliosTM Baseline
Offset function). Due to the visual nature of flow cytometry
interpretation, it is critical that both the acquiring and
analyzing operators have a full understanding of the effects
of all acquisition and analysis software functions.
P28
FREQUENCY OF CD34+ HEMATOGONE SUBSET
IN PERIPHERAL BLOOD STEM CELL APHERESIS
PRODUCTS USING THE ISHAGE PROTOCOL
Sarah L Ondrejka, Deborah Katanik, Betty Gay, Bruce
Briggs, Florence Namiotka, Lisa McTaggart, Hien Duong,
Eric D Hsi
Cleveland Clinic
Background:
CD34+ cell enumeration is a critical step in
graft harvesting for hematopoietic stem cell transplantation.
Using the ISHAGE protocol, we periodically find a separate
population of CD34+ events, suspected to consist of
hematogones (B-lymphocyte precursors) that falls within
the gating guidelines for R3 and thus is not excluded
from the total CD34+ value reported. We investigated the
frequency with which these cells occur in peripheral blood
stem cell (PBSC) apheresis products and characterized
their phenotype.
Methods:
PBSC apheresis samples
were analyzed for the presence (≥0.01%) of the additional
CD34+ population. The CD34+ subset was analyzed with
antibodies for CD19, CD20, and CD10. The proportion
of these cells relative to total CD34+ cells was compared
between allogeneic and autologous sources.
Results:
Between 1/4/2011 – 12/6/2011, 274 apheresis samples
were evaluated. 61% contained the additional CD34+
population. This population comprised a median of 7.14%
of total CD34+ cells (range 1.25 – 77.97). CD19+, CD20+/-,
and CD10+ expression pattern in a subset (=43) of positive
samples confirmed that the population corresponded to
hematogones. The CD34+ hematogone subset in allogeneic
PBSC apheresis products (median 2.99% of total CD34+
cells, range 1.29 – 33.33) was not significantly different from
autologous products (median 5.63%, range 1.25 – 16.67;
P=0.23).
Summary:
Hematogones are captured in the
ISHAGE gating protocol and may comprise a substantial
fraction of CD34+ cells in PBSC apheresis products.
Whether inclusion may adversely affect engraftment clinical
endpoints is the subject of ongoing investigation.
POSTER ABSTRACTS