Table 3.
Experimenta | Fraction Analyzed |
Volume (μL)b | RBC Count (× 106 Cells/μL) | %RBC Depleted |
%RBC Depleted Flow Cytometryc | WBC Count (× 103 Cells/μL)d | Total % WBC Recoverye | % Granulocytesf | % Monocytesg | % Lymphocytesh | % B-Cellsi | % T-Cellsj | Run Time (Mins) |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
With Optimized Buffer | |||||||||||||
1 | Input | 200 | 2.66 | – | – | 4.0 | 87 +/− 5% | 54.0 | 5.3 | 40.7 | 9.3 | 75.5 | 21.0 |
Product | 289 | 0.01 | 99.62% | 99.993% | 2.4 | 52.6 | 14.5 | 32.9 | 11.9 | 77.1 | |||
2 | Input | 200 | 2.01 | – | – | 3.2 | 88 +/− 6% | 47.6 | 9.8 | 42.8 | 9.0 | 85.9 | 16.0 |
Product | 255 | 0.00 | 100.00% | 99.974% | 2.2 | 52.2 | 10.4 | 38.2 | 10.3 | 87.3 | |||
3 | Input | 200 | 2.45 | – | – | 2.6 | 78 +/− 7% | 60.3 | 8.6 | 30.0 | 15.7 | 67.3 | 17.0 |
Product | 240 | 0.00 | 100.00% | 99.997% | 1.7 | 64.4 | 7.4 | 29.1 | 17.0 | 63.8 | |||
4 | Input | 200 | 2.23 | – | – | 4.7 | 96 +/− 4% | 60.3 | 9.3 | 30.3 | 13.0 | 79.3 | 18.0 |
Product | 281 | 0.01 | 99.55% | 99.993% | 3.2 | 61.8 | 8.2 | 28.4 | 14.6 | 74.9 | |||
5 | Input | 200 | 2.06 | – | – | 2.0 | 81 +/− 9% | 42.3 | 7.9 | 46.9 | 9.2 | 77.0 | 18.0 |
Product | 248 | 0.00 | 100.00% | 99.961% | 1.3 | 40.0 | 10.5 | 47.4 | 8.8 | 77.8 | |||
6 | Input | 200 | 2.19 | – | – | 2.1 | 98 +/− 8% | 53.6 | 7.3 | 38.4 | 9.4 | 64.3 | 20.0 |
Product | 241 | 0.00 | 100.00% | 99.991% | 1.7 | 51.2 | 8.0 | 39.8 | 9.0 | 65.6 | |||
Mean Depletion or Recovery or Run Time | 99.8 + /− 0.09% | 99.985+/− 0.006% | 88 +/− 3% | 18.3+/-0.8 | |||||||||
Mean Difference {Product vs. Input) | 0.7+/− 1.3% | 1.8 +/− 1.6% | −2.2+/− 1.4% | 1.0 +/− 0.5% | −0.5 +/− 1.1% | ||||||||
Control with BSA Buffer | |||||||||||||
1 | Input | 200 | 2.68 | – | – | 3.8 | 77 +/− 5% | 54.0 | 5.3 | 40.7 | 9.3 | 75.5 | 22.0 |
Product | 278 | 0.01 | 99.63% | 99.991% | 2.1 | 53.0 | 11.9 | 35.1 | 5.5 | 82.0 |
“Control with BSA Buffer” Experiment 1 used BSA buffer and the same donor blood sample, and was run at the same time as Experiment 1 “with Optimized Buffer”. Experiments 1 – 6 with “Optimized Buffer” used the buffer containing Polyoxamer instead of BSA.
%RBC depletion was determined using relative ratios of RBC/WBC for both the Coulter Counter and flow cytometry approaches.
Granulocytes were identified by flow cytometry as DRAQ5+ signals with characteristic high FSC and SSC.
Monocytes were identified as DRAQ5+ with characteristic intermediate FSC and SSC.
Lymphocytes were identified as DRAQ5+ with characteristic low FSC and SSC.
B cells were identified as DRAQ5+/CD19+ with FSC and SSC of lymphocytes.
T cells were identified as DRAQ5+/CD3+ with FSC and SSC of lymphocytes.