Beckman Coulter IM3632 Manual De Instrucciones página 125

APPENDIX
- EXAMPLES
The following eight histograms correspond to the analysis of the Stem-Trol Control Cells stained with Stem-Kit
Reagents. Below are histograms obtained on a COULTER EPICS XL/XL-MCL flow cytometer equipped with
System II Software (Version 3.0).
Histogram 1: displays all events minus all Stem-Count Fluorospheres (i.e. "-H" see Histogram 5).
Notes:
Position Region A to include all CD45 events (leukocytes) while excluding CD45- debris. Position Region E
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to include only lymphocytes (bright CD45, low Side Scatter). Region A is intended to serve as an appropriate
denominator (total WBC) in the calculation of the percentage of CD34 HPC.
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Histogram 2: displays events from region A.
Adjust Region B to surround CD34 Stem-Trol Control Cells, including CD34dim events. Upper limit along the
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y-axis may be set to include CD34 events with intermediate Side Scatter for CD34 Stem-Trol Control Cells
+ +
Histogram 3: displays events from A and B.
Adjust Region C to include cells forming a cluster with characteristic CD34 Stem-Trol Control Cells (i.e.
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intermediate Side Scatter and low to intermediate CD45 staining). It is the cluster of events that determines where
Region C is centered. Brightly FITC stained events (platelet aggregates and mature monomyeloid cells) must be
excluded in the setting of this region.
Histogram 4: displays events from regions A and B and C.
Region D must include events characteristic of Stem-Trol Control Cells. Once the analysis is done, the absolute
count of total CD34 Stem-Trol is given on the statistic printout related to the CD34 Stem-Trol (Region D).
+ +
Histogram 5: displays all events.
Important: This histogram is useful to visualize the lower limit of CD45 expression within the CD34+ events.
Set Quadstat Region I2 to enclose all CD45+ CD34 events. Set the left boundary of Quadstat Region I2 to include
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all CD34 CD45dim events. Verify on Histogram 1 that the lower limit of Region A includes all CD34+ events (use
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Histogram 5 as a guide).
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