ACQUISITION RATES EFFECT ON MULTIPARAMETRIC RARE EVENT ANALYSIS:
AN INSTRUMENT COMPARISON
Program Number 164
John Tigges, Vasilis Toxavidis, Heidi Mariani
Beth Israel Deaconess Medical Center/Harvard Stem Cell Institute
3 Blackfan Circle
Boston, MA 02215
DsRed TRegs
RESULTS
INTRODUCTION
• Separation, and resolution show noticeable differences. Major discrepancy on Thy1.1 Tregs.
 DsRed TRegsGallios avg. % gated=6.44
LSR II avg. % gated=6.45
Difference=0%
 Thy1.1 TRegsGallios avg. % gated=10.48 LSR II avg. % gated=6.72
Difference=36%
 LT HSCGallios avg. % gated=30.21 LSR II avg. % gated=28.68
Difference=5%
• MFI is significantly better on the Gallios. This is true across the board of variable speeds.
 DsRed TRegsGallios avg. MFI=41.38
LSR II avg. MFI=37.30
Difference=10%
 Thy1.1 TRegsGallios avg. MFI=26.96
LSR II avg. MFI=24.77
Difference=8%
 LT HSCGallios avg. MFI=27.76
LSR II avg. MFI=18.64
Difference=33%
The current paradigm in Flow Cytometry is the increasing number of laser
lines, multiparameter instrument capabilities, and increasing number of
antibodies; flow cytometric applications have become more complex and
with greater variability. One of the major challenges pertaining to
instrumentation is dealing with reliability of the optics and the laser lines.
It is of extreme importance to be able to shape the laser beams elliptically,
focus them to the cellular interrogation point, and not lose laser power
during this process. In conjunction with lasers and optics, the fluidics
system plays an integral part in obtaining the best data possible from the
cellular preparation. Event rate, determined by amount of pressure applied
to the sample, rate of fluid through the system, and cellular concentration,
is the driving force behind obtaining reproducible and reliable data.
However, with advances in optical alignment and fluidic (hydrodynamic)
focusing, even rare event analysis can be obtained at varying speeds
without compromising data efficiency.
• Average difference for % gated is 13.67% and average difference for MFI is 17%.
• Median % Total difference is approximately 50% (Both Thy1.1 TRegs and LT HSC).
• The bar graphs below demonstrate differences amongst % Total, % Gated, and MFI at the varying
speeds for all sample types.
Thy 1.1 TRegs
MATERIALS AND METHODS
DsRed TRegs
Thy1.1 TRegs
LT HSC
30
50
40
45
35
25
40
30
35
20
Hematopoietic Stem Cells (HSC) from Whole Bone Marrow and
Regulatory T Cells were analyzed at varying speeds with differing
numbers of parameters used and on two commercially available FACS
analyzers. HSCs were labeled with FITC, PE-Cy7, APC, APC-Cy7, and
Pacific Blue; TRegs were labeled with GFP, DSRed, and PE-Cy7; and Thy
1.1 TRegs were labeled with FITC, PE, APC, APC-Cy7, and DAPI. Cells
were then acquired on both a Becton Dickinson SORP LSRII equipped
with a 561nm laser and a Beckman Coulter Gallios at speeds of 5K, 10K,
15K, 20K, and 25K for Tregs.
25
30
25
20
15
20
15
10
15
10
10
5
5
5
0
5K
5K
10K 10K 15K 15K 20K 20K 25K 25K
Gallios LSRII Gallios LSRII Gallios LSRII Gallios LSRII Gallios LSRII
0
0
5K
Gallios
5K
LSRII
10K
Gallios
% Total FL-5
The SORP LSR II is equipped as follows: 488nm- FITC 530/30; 561nmPE 585/15, DsRed 585/15, PE Cy7 780/60; 640nm- APC 670/14, APC
Cy7 780/60; 405nm- Pacific Blue 450/50; UV- DAPI 450/50.
The Gallios is equipped as follows: 488nm- FITC 525BP, PE 575BP, PE
Cy7 755LP, DsRed 575BP (acquired using the PE channel; Gallios is not
equipped with a 561nm); 633nm- APC 660/20, APC Cy7 755LP; 405nmPacific Blue 450/40, DAPI 450/40 (acquired using Pacific Blue channel
with violet excitation).
Both instruments underwent initial quality check according to
manufacturer’s guidelines (Becton Dickinson- Cytometer Set-up and
Tracking Beads; Beckman Coulter- Flow Check Pro Beads). Instrument
performance was then verified using Spherotech 8 peak rainbow beads.
Samples were acquired on High sample rate, manufacturer’s preset
threshold off FSC (Gallios=100; LSRII=5000), and 10,000 Total events.
Samples were analyzed with Beckman Coulter’s Kaluza 1.0 Software. All
plots and gates remained consistent for each sample type.
10K
LSRII
15K
Gallios
% Gated FL-5
15K
LSRII
20K
Gallios
20K
LSRII
5K
5K
10K
10K
15K
15K
20K
20K
Gallios LSRII Gallios LSRII Gallios LSRII Gallios LSRII
X-Mean FL-5
CONCLUSIONS
Long Term HSCs
In both cases, no major intra-instrument variation due to speed was apparent in
cellular number, % total, % gated or Mean Fluorescent Intensity. However, most
notably, differences were viewed inter-instrument in % Total, % Gated, and MFI.
This was particularly noticeable at the higher speeds analyzing the rare event
populations (% Total ≤ 2). Overall separation and resolution were significantly
different independent of speed.
While an instrument can show consistency and reliability throughout variable
event rate, it is not an obvious indicator of an instrument’s overall performance.
The ability of an instrument to deliver a sample to the interrogation point
effectively without any loss of laser intensity at that point or to the PMT is crucial
to data acquisition and analysis. This is abundantly clear in rare event analysis,
where cells are minimal and any loss of signal is detrimental. We have shown
this to be true of the Gallios system with its patented Boulevard technology and
enhanced fluidics system.
For additional information please contact:
John Tigges
Flow Cytometry Core
Beth Israel Deaconess Medical Center
jtigges@bidmc.harvard.edu