Analyze - MITweb.mit.edu/flowcytometry/www/BD-Horizon-Tour-2016...single-positive populations. • Although the spread of BV650 is equivalent into BV605 & BUV737, it has less of an
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AnalyzeA step-by-step approach to build and analyze a multicolor panel
The BD Horizon™ Global Tour | 1
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Alexa Fluor® is a registered trademark of Life Technologies Corporation.
Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University and are made and sold under licensefrom GE Healthcare only for research and in vitro diagnostic use. Any other use requiresa commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
Trademarks are the property of their respective owners.
Fluorescence spillover and spread impact resolution
• Understanding the impact of fluorescence spillover and spread is the key to good panel design.
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CD
56
CD3CD3
CD
56
Spread impacts resolutionSpillover introduces spread and background
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Spillover introduces background and spread
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Amount of spillover
The amount of the spread
SOV =
9.15%
PE-Cy 5
PE
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Amount of spillover
The amount of the spread
SOV =
0.86%
FITC
PE
Spillover introduces background and spread
Spillover introduces background and spread
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Amount of spillover
The amount of the spread
SOV =
26.40%
PE-CF594
PE
Spillover introduces background and spread
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The amount of the spread
Amount of spillover
MFI =
Reagent brightness
420
35,000
7,000
1,500PE
PE-CF594
• Population resolution for the PE-CF594 fluorescence parameter is decreased by increased spread due to PE spillover from another fluorescence parameter.
• To maximize the resolution of a given double-positive subpopulation:
– Minimize fluorescence spread into the detector that defines that population
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Amount of spillover
The amount of the spread
PE-CF594
PE
Antigen density( Fluorochrome
brightness )
Spillover introduces background and spread
Spillover Values (SOVs)
• Are totally dependent upon gain settings (PMTVs).
• Does not always accurately reflect the impact of spread.
Spread impacts the resolution of double-positive populations
BV
60
5
• Spread is most important when considering reagents for antigens co-expressed on a subpopulation.– Double-positive populations
need to be resolved from the single-positive populations.
• Although the spread of BV650 is equivalent into BV605 & BUV737, it has less of an impact on the resolution of a marker stained with BUV737.
• Although the spread of PE is equivalent into PerCP-Cy™5.5 and PE-CF594, it has less of an impact on the resolution of a marker stained with PE-CF594.
CD4 BV650 CD4 BV650
BU
V7
37
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Resolution of double-positive populations
A+
CDB (PE)
A+B+
A-
A-B+
• Here is a classic resolution of a single-positive (A+) population being resolved from a negative (A-) population.
• Adding a second co-expressed marker, we now have to resolve a double-positive (A+B+) population from a single (A-B+) population.
– Resolution measured in Stain Index (SI)
– Resolution measured in a double-positive Stain Index (DP-SI)
Loss ofResolution
• The spread of fluorochrome B into the A detector reduces the resolution of a double-positive (A+B+) population from the A-
B+ population.
CD
A(P
E-C
F5
94
)
SI700
DP-SI50
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Primary
Fluor FITC PE
PerCP-
Cy5.5 PE-Cy7 APC
APC-
H7
FITC 68 61 70 74 67 72
PE 170 340 350 216 358 356
PerCP-Cy5.5 73 15 131 60 131 124
PE-Cy7 454 132 63 522 375 265
APC 522 471 169 444 486 287
APC-H7 120 127 42 15 18 116
Double-Positive Stain Index*Co-expressed Fluor
Double-positive stain index matrix
• This table shows double-positive
Stain Index values
– A double-positive Stain Index less than
the single-positive Stain Index indicates
that the spread of the secondary co-
expressed fluorochrome has reduced
resolution.
* Run on a BD FACSVerse™ flow cytometer
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Primary
Fluor FITC PE
PerCP-
Cy5.5 PE-Cy7 APC
APC-
H7
FITC 68 61 70 74 67 72
PE 170 340 350 216 358 356
PerCP-Cy5.5 73 15 131 60 131 124
PE-Cy7 454 132 63 522 375 265
APC 522 471 169 444 486 287
APC-H7 120 127 42 15 18 116
Double-Positive Stain Index*Co-expressed Fluor
Double-positive stain index matrix
• This table shows double-positive
Stain Index values
– A double-positive Stain Index less than
the single-positive Stain Index indicates
that the spread of the secondary co-
expressed fluorochrome has reduced
resolution.
• This table shows the relative loss of
resolution which can be evaluated as the
percent difference between the Stain Index
and the double-positive Stain Index
– 1- (50/700) x 100%.* Run on a BD FACSVerse
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Primary
Fluor FITC PE
PerCP-
Cy5.5 PE-Cy7 APC
APC-
H7
FITC 68
PE 340
PerCP-Cy5.5 131
PE-Cy7 522
APC 486
APC-H7 116
Resolution Impact Matrix*Co-expressed Fluor
The resolution impact matrix
SI (Primary)
<20% 20-40%
% loss of SP-SI40-60% 60-80% >80%
• Color coding the percent loss makes it
easy to visualize which fluorochromes
have the greatest impact on each
other.
• This table shows double-positive
Stain Index values
– A double-positive Stain Index less than
the single-positive Stain Index indicates
that the spread of the secondary co-
expressed fluorochrome has reduced
resolution.
• This table shows the relative loss of
resolution which can be evaluated as
the percent difference between the
Stain Index and the double-positive
Stain Index
– 1- (50/700) x 100%.
* Run on a BD FACSVerse
The BD Horizon™ Global Tour | 17
Primary
Fluor FITC PE
PerCP-
Cy5.5 PE-Cy7 APC
APC-
H7
FITC 68
PE 340
PerCP-Cy5.5 131
PE-Cy7 522
APC 486
APC-H7 116
Resolution Impact Matrix*Co-expressed Fluor
Using the resolution impact matrix
• The resolution impact matrix provides a quick visual tool to help assess
potential problems with spread when evaluating potential use of two
fluorochromes for co-expressed markers on a population of cells.