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When at least 1,000 fluorospheres singlets are acquired, the absolute count for the CD34
+
Stem-Trol Control Cells
is automatically adjusted and can be taken directly from Region D statistics printout results.
Table (below):
Example of statistics obtained with tube TROL 45/34/7-AAD on System II Software, using the CAL FACTOR
function, where:
•
1,024 is the Stem-Count Fluorospheres Assayed Concentration.
•
5,248 is the total number of fluorospheres accumulated in the CAL region during the full acquisition time.
In this example, all counts are adjusted by: 1,024/5,248.
Stats: Not
Normalized Hist
Listgating: Disabled
Region ID
Counts Adjusted
(Cells/µL)
1
A
LEUKS
9,798
2
B
CD34 POS
279
3
C
CD45 DIM
264
4
D
Stem-Trol Control
Cells
248
7
G
CAL 1,024
1,024
5
H
All Stem-Count
Fluorospheres
1,046
1. Record the adjusted absolute counts (cells/µL) in Regions D from Tubes TROL 45/34/7-AAD.
.
Example:
.
Tube TROL 45/34/7-AAD: Stem-Trol Control Cells = 248 cells/µL.
2. Multiply this precise count by the normalization factor (N) = 5.
.
NOTE:
.
It is necessary to normalize the absolute count of Stem-Trol Control Cells with regard to the added volumes during the test:
Determine N by dividing the volume of the counting reference (Stem-Count Fluorospheres singlets), by the volume of
Stem-Trol Control Cells added to the test tube.
.
..
Example:
.
100 µL Stem-Count Fluorospheres are added to 20 µL of Stem-Trol Control Cells:
.
N = 100 ÷ 20 = 5
.
Normalized Stem-Trol Control Cells absolute count = 248 x 5 = 1,240 cells/µL.
3. Verify the Stem-Trol Control Cells absolute count obtained by comparing it to the Stem-Trol Control Cells.
Assayed Concentration (cells/µL) stated on the vial label. The Stem-Trol Control Cells absolute count obtained
must be within ±15% of the Stem-Trol Control Cells Assayed Concentration. The staining method and lysing
process are now validated.
.
Example:
.
Stem-Trol Control Cells Assayed Concentration = 1,380 cells/µL.
.
Range of accepted values: 1,173 – 1,587 (1,380 ± 207) cells/µL
.
The normalized absolute count = 1,240 cells/µL is within the range.
.
..
IMPORTANT:
.
If the Stem-Trol Control Cells absolute count falls outside of the range of accepted values, determine whether the
pipetting has been correctly performed, especially for the Stem-Count or Flow-Count Fluorospheres and Stem-Trol
Control Cells. Visually check the flow cytometer reports and the quality of staining and lysing process in your test tubes.
Verify the way the region boundaries were set. If necessary, repeat the preparation with new test tubes. Acquire and
analyze both the new and the previous series of tubes. Compare the two results. If there is no agreement, call your local
Beckman Coulter Representative.
