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Following the application of the BD FACSlyse the eosinophils are easiest
detected in log sidescatter vs autofluorescence or their position in the log
side scatter versus antibody fluorescence plot (see attached word97 document,
data from the Beckmann Coulter XL). As the signal intensity on neutrophils and
eosinophils correlates with the volume, the 2 dimensional region is the more
accurate analysis method. We use the same method for adhesion molecule analysis
on granulocytes. For a histogram based analysis one would have to transform the
axis for optimal separation as done already some years ago by the 'hidden line'
feature in the software from Partec. If you set your background region for a
higher percentage you will also see a difference in the subtracted % positives,
but still never get as good ad 2D.
Regards
Gerhard
-----Original Message-----
From: Becky Kelly [SMTP:Eak@medicine.wisc.edu]
Sent: Tuesday, May 23, 2000 12:55 AM
To: Cytometry Mailing List
Subject: analysis question
I am interested in opinions from this esteemed group concerning the best way to
analyze the following data (see attachment in MSWord format). My colleagues
and I can not reach a consensus. We are assessing cell surface determinants on
eosinophils in lysed whole blood. Since lymphocytes are also analyzed,
compensation is set based on FITC-CD3/PE-CD19. An eosinophil gate is
established by back-gating on CD45bright/CD14 dim (autofluorescent) cells and
a CD16/CD14 cocktail is used to exclude any neutrophils or monocytes that might
contaminate the gate. To determine the % positive cells, we are using either
the dot plot or histogram. For dot plot, a tight gate (R2) is set around
autofluorescent cells in the PE-IgG control sample (Top left panel). A larger
gate (R3) is established to allow identification of positive cells. The %
positive cells are calculated as (1-(R2/R3))X100. Histograms are gated on R1
(eosinophils on scatter plot) and R3. M1 is set on isotype control. For
signals that have a large shift (ie. CD11b, middle panel), the % positive are
the same using either method. When the signal is weak, such as for cytokine
receptors (right panel), only a small proportion (in this case 27%) are
positive based on histogram; whereas, the dot plot would indicate nearly 100%
positive based on a shift out of the "negative" region. Any opinions, comments
about how to express these data? Is there any reason why the dot plot method
would not be acceptable?
<< File: flow eos question.doc >>
--33364686-6808-959881400=:87
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Content-Language: en-GBR
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\pard Following the application of the BD FACSlyse the eosinophils are easiest detected
in log sidescatter vs autofluorescence or their position in the log side scatter versus
antibody fluorescence plot (see attached word97 document, data from the Beckmann Coulter
XL). As the signal intensity on neutrophils and eosinophils correlates with the volume,
the 2 dimensional region is the more accurate analysis method. We use the same method
for adhesion molecule analysis on granulocytes. For a histogram based analysis one would
have to transform the axis for optimal separation as done already some years ago by the
'hidden line' feature in the software from Partec. If you set your background region
for a higher percentage you will also see a difference in the subtracted % positives,
but still never get as good ad 2D.\par
Regards\par
Gerhard\par
\par
\objattph\'20\par
\pard\par
\cf0\protect\f1\fs16 -----Original Message-----\par
\protect0\pard\protect\fi-1440\li1440\tx1440\b From:\tab\b0 Becky Kelly
[SMTP:Eak@medicine.wisc.edu]\par
\b Sent:\tab\b0 Tuesday, May 23, 2000 12:55 AM\par
\b To:\tab\b0 Cytometry Mailing List\par
\b Subject:\tab\b0 analysis question\par
\protect0\pard\protect\lang2057\f2\fs20\par
I am interested in opinions from this esteemed group concerning the best way to
analyze the following data (see attachment in MSWord format). My colleagues and I can
not reach a consensus. We are assessing cell surface determinants on eosinophils in
lysed whole blood. Since lymphocytes are also analyzed, compensation is set based on
FITC-CD3/PE-CD19. An eosinophil gate is established by back-gating on CD45bright/CD14
dim (autofluorescent) cells and a CD16/CD14 cocktail is used to exclude any neutrophils
or monocytes that might contaminate the gate. To determine the % positive cells, we
are using either the dot plot or histogram. For dot plot, a tight gate (R2) is set
around autofluorescent cells in the PE-IgG control sample (Top left panel). A larger
gate (R3) is established to allow identification of positive cells. The % positive
cells are calculated as (1-(R2/R3))X100. Histograms are gated on R1 (eosinophils on
scatter plot) and R3. M1 is set on isotype control. For signals that have a large
shift (ie. CD11b, middle panel), the % positive are the same using either method.
When the signal is weak, such as for cytokine receptors (right panel), only a small
proportion (in this case 27%) are positive based on histogram; whereas, the dot plot
would indicate nearly 100% positive based on a shift out of the "negative" region.
Any opinions, comments about how to express these data? Is there any reason why the
dot plot method would not be acceptable?\par
\par
<< File: flow eos question.doc >> \par
}
--33364686-6808-959881400=:87--
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