1. Instrument QC and Qualification

2. Overview Why QC is Important LSRII Optical Configuration LSRII QC
Validation
Optimization
Calibration
Standardization
Practice Analysis

3. Characterizing the Cytometer

4. Challenges…
Instrument - optical configuration, optimization, standardization, and calibration
Reagent - optimization and standardization
Sample processing
Staining protocols
Data Analysis - compensation & gating
Operators
Volume of data (death-by-excel!)
More on instruments - configuration = laser (power and wavelength… ex percp is great w/ low power but crap with higher power, and PE tandems are better excited using green, rather than blue (yet another mario/perfetto pub). Optimization is hitting voltage sweet-spots for maximum signal:noise and minimal interference (another perfetto pub). Calibration is target channels (where positive stays in same place across assays, instruments, labs, etc)
Note on reagents - tandems are very delicate; when handled properly very nice, but exposure to light/certain fixatives/ambient temperature may result in false positives, generated by degradation of the dye (ex CD8 APC-Cy7 and IFNg APC, if APC-Cy7 tandem degrades, then a small amount of CD8 APC signal will appear as IFNg APC). So, vigilance in following protocols is key.
Changed preparation to processing (as in PBMC processing), so as to distinguish from staining protocols
I don’t understand “waiting time” but left it as is
Gating is the largest source of variation, AFTER the areas listed above have been addressed…holden et al BMC Immunology manuscript example for ICS would win browny points for Holden fans in the audience ;)
Duke University Medical Center

5. Two Methods of Instrument Charaterization
BD CS&T: Cytometer Setup and Tracking

6. Instrument Performance
Parameters CS&T Perfetto
Laser (amps vs mW) yes yes
CV (resolution) yes (target) yes (range)
Signal-to-noise ratio NO yes (range)
Linearity Yes (range) yes (range)
Specific fluorescence NO yes (target)
Automated YES NO

7. Each Experiment & Troubleshooting
Instrument QC
Frequency
Purpose
Initial Characterization & After Optical Service
Validation: optimal voltage range for each detector
Once Per Assay
Optimization: assay specific optimal voltage for each detector & assay specific target channels
Each Experiment & Troubleshooting
Calibration: set detectors to assay specific target channel values for specimen acquisition
Standardization: Monitor trendlines using assay specific target channels over time

8. Key Performance Factors in High-Quality Flow Cytometry Data
Resolution of subpopulations, including dim subpopulations
Sensitivity
Relative measured values of fluorescence
Linearity and accuracy
Reproducibility of results and cytometer performance
Tracking
Comparison of results across time and amongst laboratories
Standardization

9. LSRII qualification Validation Linearity Resolution (CV)
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization
Select Optimal Voltages for Inst Performance with Assay-Specific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)
Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision
Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trendlines

10. Linearity Definition: Proportionality of output to input
Method: Access linearity using the ratio of two pulses over voltage range of the PMT
Significance: Linearity is important for compensation
(Median Pk6 - Median Pk5)
Median Pk5
= Constant

11. Linearity Important for fluorescence compensation
Defined as proportionality of output (MFI) to input (Fluorescence/ # of photons)
Important for fluorescence compensation
Compensation of data in the last decade involves subtraction of large numbers
Small errors (non-linearity) in one or both large numbers can cause a large absolute error in the result
Ab = X
Ab = 2X
X Abs
2X Abs =
1000 MFI
2000 MFI
1000
2000
3000

13. Linearity: Effect on Compensation
Compensation of data in the last decade involves subtraction of large numbers
Errors (non-linearity) in one or both large numbers can cause a large absolute error in the result
FITC PE A 68 80
Detector
Median Fluorescence Intensity (MFI)
5921 79 C
1796 75 B
73,000
365 D
BD CompBeads stained with varying levels of FITC-Ab.
Compensation was set using samples A and C.
This cytometer had a 2% deviation from linearity above 50,000 units.

14. LSRII Qualification Validation Optimization Calibration (“Daily QC”)
Linearity
Resolution (CV)
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization
Select Optimal Voltages for Inst Performance with Assay-Specific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)
Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision
Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines

15. cvs

16. Instrument Sensitivity: Two Definitions
Defining sensitivity
Threshold: Degree to which a flow cytometer can distinguish particles dimly stained from a particle-free background. Usually used to distinguish populations on the basis of Molecules of Soluble Equivalent Fluorochrome (MESF).
Resolution: Degree to which a flow cytometer can distinguish unstained from dimly stained populations in a mixture.
How to measure instrument-dependent sensitivity?
Resolution sensitivity is a function of three independent instrument factors: Br Qr
Electronic noise (SDen)
Although low MESF values indicate “good” instrumental (threshold) sensitivity, this by no means guarantees an instrument will be able to resolve dim over background.

17. Background contributions

18. Why is Br important? High Br widens negative and dim populations.
High Br value = lower resolution
Low Br value = higher resolution
Low Br
High Br

19. Br: Optical Background from Propidium Iodide
Example: It is common to use propidium iodide (PI) to distinguish live from dead cells. Propidium iodide was added in increasing amounts to the buffer containing beads, and Qr and Br were estimated.
PerCP
1000
2000
3000
4000
5000
6000
7000
8000
9000 1 2 3 4 5 6
PI-free dye (µg) Br
0.01
0.02
0.03
0.04
0.05 Qr
Residual PI in your sample tube will increase Br, which will reduce sensitivity.

21.    Relative Q: Qr # photoelectrons Qr # fluorescence molecules
Qr is photoelectrons per fluorescence unit and indicates how bright a reagent will appear on the sample when measured in a specific detector.
# photoelectrons Qr =
# fluorescence molecules
PMT 1 
2 photoelectrons 
Qr =
= 0.25
8 fluorescence molecules
In the example below, a cell with a the same number of fluorescence molecules can be detected differently by two different systems. A system with a high Qr would detect a larger number of photons for the same population than a system with a lower Qr. If a system has a lower relative Q, it produces fewer photoelectrons per fluorescence molecule, which would contribute to a higher CV and lower resolution sensitivity.
The CS&T application calculates Qr for all fluorescence parameters based on the median and the rCV values for each of the CS&T fluorescent beads.
PMT 2
1 photoelectron 
Qr =
= 0.125
8 fluorescence molecules

22. Low Qr value = lower resolution High Qr value = higher resolution
Why is Qr important?
A system with a higher Qr has a better resolution than a system with a lower Qr.
Low Qr value = lower resolution
High Qr value = higher resolution
High Qr
Low Qr

23. What Factors Affect Qr? Laser power Optical efficiency
PMT sensitivity (red spectrum)
Poor PMT performance
Dirty flow cell
Dirty or degraded filter
Qr is affected by the laser power, which in turn is affected by laser alignment. Diminished laser power can also indicate a dying laser.
It is also affected by the overall optical efficiency and PMT spectral sensitivity. The PMTs are less sensitive in the red spectrum. A lower Qr reading is usually observed in the red parameters.
Optical design also has an impact on the relative Q. A low reading can result from any of the following:
dirty flow cell
dirty or degraded filter
poor PMT performance

24. Spillover Decreases Resolution Sensitivity
Spread from APC Cy-7 background
Population resolution for a given fluorescence parameter is decreased by increased spread due to spillover from other fluorochromes.

25. Summary: Instrument Performance and Sensitivity
Qr and Br are independent variables, but both affect sensitivity.
Increases in Br or decreases in Qr can reduce sensitivity and the ability to resolve dim populations.
On digital instruments, BD FACSDiva™ software v6 and CS&T provides the capability to track performance data for all of these metrics, also allowing users to compare performance between instruments. Br Qr y
Sensitivit
relative
Instrument performance can have a significant impact on the performance of an assay.

26. Resolution vs. Background
Negative
Population
Positive
Negative population has
low background
Populations well resolved
Negative population has
high background
Populations not resolved
Negative population has
low background
high CV (spread)
Populations not resolved
The ability to resolve populations is a function of both background and spread of the negative population.

27. Measuring Sensitivity: The Stain Index
The Stain Index is a measure of reagent performance on a specific cytometer, a normalized signal over background metric.
Brightness
Width of negative
The brightness is a function of the assay (antigen density, fluorochrome used).
The width of the negative is a function of:
Instrument performance (Qr, Br, and SDen) [single-color]
The assay
(Fluorescence spillover / compensation) [multicolor]
The cell population

28. Stain Index: Normalized Signal/Background
D/W = Relative Brightness
(SI)
Index
Stain =
Goal: Normalize the signal
to the spread of background
where background may be
autofluorescence, unstained
cells, or compensated cells
from another dye dimension. D
Wunstained
Wcompensated dye
Stain Index… a better way to determine relative brightness for individual fluors.
D = difference between positive and negative peak medians
W = the spread of the background peak (= 2X rSDnegative)
1Stain Index: metric used by Dave Parks, Stanford – Presented at ISAC 2004

29. Electronic Noise (SDEN): Determining Baseline PMT Voltages
The BD FACSDiva™ 6.0 CS&T module analyzes dim particles, which are similar to dim cells’ brightness, allowing relevant detector baselines to be visualized by plotting fluorescence intensity vs (PMT gain, CV, and SD)
PE: Detailed Performance Plot
Dim Bead
For this detector, the SDEN = 18
Fluorescence intensity of dim bead = 10 x SDEN = 180 CV
Standard Deviation
PMT Voltage
10000
1000
Determine PMT voltage required to set the dim beads at = 500 volts = baseline voltage
500 V
1000
CV or SD
PMT Voltage
As PMT voltage is lowered, CV increases  resolution decreases
100 18
As PMT voltage is increased CV unchanged  resolution unchanged 10
100
180 1 10
100
1000
10000
100000
Median Fluorescence Intensity

30. PMT Voltages: Optimal Gains Can Reduce Classification Errors
CD4 dim monocytes
CD4+ lymphocytes
CD4 negative
% Negative in CD4+ Monocyte Gate
0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
12.0%
-100
100
PMT Voltage Offset
% Negative in CD4 Gate
650 V
750 V

32. Methods Used for Validation
Which Beads to Use
What Values to Plot
Selection Criteria

33. Example LSRII Optical Configuration
Blue Laser
(488nm)
Duke LSRII
SSC
Blue
FITC
515/20
505LP
575/25
550LP PE ﾐ
488LP
Red Laser
(635nm)
Duke LSRII
APC
Alexa
680
710/50
685LP
780/60
740LP
Cy7
660/20
Green Laser
(532nm)
Duke LSRII PE
Cy7
Cy5 TR
Cy5.5
PE-
Green
780/40
610/20
710/50
660/40
575/25
740LP
640LP
600LP
690LP
Violet Laser
(407nm)
Duke LSR II
QDot
705
605
545
585
655
565
705/70
585/42
660/40
605/40
560/40
670LP
595LP
570LP
630LP
557LP
535LP Am
Cyan
515/20
505LP
Cas
Blue
450/50
Change to config from Jennifer
Duke University Medical Center

34. Beads Used for LSRII Validation
8 Peak Rainbow: v in 50v increments (all PMTs)
Non-fluorescent to Very Bright; Broad Spectrum Ex & Em
8pks
Run: Once, then after optical service - “High” flow rate & LOG scale
Uses: Validation
Check Linearity: (MedianPk6-MedianPk5)/MedianPk5
Check CV: CV Pk5
Check S:N (LLOD & LLOQ): MedianPk5/MedianBlank
Unstained Comp Beads: v in 50v increments (all PMTs)
Non-fluorescent
1pk
Run: Once, then after optical service- “High” flow rate & LOG scale

35. Example of Optimal PMT Performance
300 Volts
350 Volts
400 Volts
450 Volts
500 Volts
550 Volts
600 Volts
650 Volts
700 Volts
750 Volts

36. Criteria for the Selection of Voltage Ranges
Lowest CV possible
Lowest Voltage possible
Highest MFI possible
Lowest background possible

37. Example of Optimal PMT Performance & Voltage Selection Criteria (Green B)
Linear
Lowest CV
Highest S:N
Lowest Voltage
Ratio = M1/B
Linearity = (M2-M1)/M1
Optimal Voltages:

38. Red A (APC-Cy7): Before (30Mar06) & After (10Apr06) Replacing PMT
30 March 2006
10 April 2006
Optimal Voltages:
Optimal Voltages: Indeterminate
High CV’s; poor S:N

39. Faulty PMT on install…
650 Volts
Before
After

40. LSRII Qualification Validation Optimization Calibration (“Daily QC”)
Linearity
Resolution
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization
Select Optimal Voltages for Inst Performance with Assay-Specific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)
Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision
Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines

41. Beads Used for LSRII Optimization
STAINED Comp Beads: validated range in 50v increments (each PMT)
Assay Specific fluorescence
1pk
Run: Once, then after optical service- “High” flow rate & LOG scale
Uses: Optimization
Optimize PRIMARY Fluorescence (Primary>Secondary)
Unstained Comp Beads: optimized voltages (all PMTs)
Non-fluorescent
Uses: Validation
Check S:N (LLOD & LLOQ)
1x or Midrange Rainbow: optimized voltages (all PMTs)
Moderate fluorescence, near cellular expression; Broad Spectrum Ex & Em
Run: Once (after optimization) - “High” flow rate & LOG scale
Establish Target Channels Linearity: medians = assay specific target channels

42. Criteria for the Selection Optimal PMT Voltages
The primary fluorescence should be the highest in the respective detector relative to all secondary detectors.
Once the ranges are established, voltages are determined based on specific FL. Each detector records a COMP bead profile, which is unique to that detector.

43. Green A: TNF PE-Cy7 460v Baseline & Final

44. Green B: CD8 PerCP-Cy5.5 450v Baseline

45. Green B: CD8 PerCP-Cy5.5 550v Final

46. Green C: CD27 PE-Cy5 350v Baseline

47. Green C: CD27 PE-Cy5 450v Final

48. Green D: CD45RO PE-TR 430v Baseline & Final

49. Green E: MIP1b PE 350v Baseline & Final

50. Duke LSRII Optimization of Specific Fluorescence
Blue
Violet
Primary Fluorescence
Red
Secondary
Fluorescence
Green

51. Neg Comp Beads

52. LSRII Qualification Validation Optimization Calibration (“Daily QC”)
Linearity
Resolution
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization
Select Optimal Voltages for Inst Performance with Assay-Specific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)
Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision
Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines

53. 1x (Target Channel Values)

54. Target Channels & Ranges

55. LSRII Qualification Validation Optimization Calibration (“Daily QC”)
Linearity
Resolution
Signal-to-Noise Ratio (S:N) - LLOD & LLOQ
Optimization
Select Optimal Voltages for Inst Performance with Assay-Specific Reagents - reduce spillover (Specificity)
Establish Assay-Specific Target Channels
Calibration (“Daily QC”)
Set Daily Voltages
Verify Voltages are within acceptable Range (P/F)
Verify CVs are within acceptable Range (P/F)
Set Target Channels for Specimen Acquisition (P/F)
Standardization - Reproducibility/Precision
Record Daily Target Channel Values
Record Daily Voltages
Record Daily CVs
Calculate Daily S:N
Plot & Review Monthly Trend lines
Duke University Medical Center

56. Daily Calibration Mid-Range or “1x” Rainbow Beads: TC settings
Moderate fluorescence, similar to cells; Broad Spectrum Ex & Em
1pk
Run: Morning & Before each Exp - “High” flow rate & LOG scale
Uses: Calibration & Standardization
Set Assay-Specific Target Channels (TCs)
Determine Daily Voltage Settings
Daily QC: P/F (CVs, voltages, trends)
LSRII Performance Verification for Exp Run: P/F (CVs & TCs)
8 Peak Rainbow & Unstained Comp Beads: TC Settings
Non-fluorescent to Very Bright; Broad Spectrum Ex & Em
8pk and 1pk
Run: Once Daily - “High” flow rate & LOG scale
Uses: Standardization
Check Linearity
Check S:N (LLOD & LLOQ)
Check Specificity
Check Resolution
Ultra Rainbow Beads: Mean ch ~150,000
VERY Bright; Broad Spectrum Ex & Em
Run: Once Daily - “Low” flow rate & linear scale
Uses: Manufacturer Recommended
Daily QC: P/F (CV & voltages)
Check/Adjust Area Scaling
Check/Adjust Time Delay
Check/Adjust Window Extension
Duke University Medical Center

57. Daily Standardization
Mid-Range or “1x” Rainbow Beads:
Record Daily For each PMT & Scatter CV HV
Median
Plot Monthly For each PMT & Scatter
HV vs Median
8 Peak Rainbow & Unstained Comp Beads:
Neg Comp Beads
Peak 5
Peak 6
Frequency
CV: peak 5
Linearity: Medianpk6-Medianpk5/Medianpk5
Signal:Noise: Medianpk5/MedianNeg
Q & B???
Ultra Rainbow Beads:
Duke University Medical Center

58. 1x Blue B Trend line
Duke University Medical Center

59. Ratio and Voltage Variation
The lower ratio at an constant CV could indicate an increase in noise of the PMT, which doe not effect the height of the signal but does effect the S/B ratio and thus the sensitivity of the detector.
Duke University Medical Center

60. LASERs have a limited lifespan…

61. Overview Why QC is Important LSRII Optical Configuration LSRII QC
LSRII Validation
LSRII Optimization
LSRII Calibration
LSRII Standardization
Practice Analysis??
Duke University Medical Center

62. Example: Stain Index, Qr, and Br Across Laboratories
Example : FITC Channel
Lab SI Qr Br 1
77.4
0.015
233 2
134.5
0.028
216 3
55.6
976 4
80.5
0.01
298 5 66
613 6
13.7
0.007
2322 7
16.2
0.018
2768
8 peak beads
High SI = Good Resolution; correlates with high Qr and low Br
Regarding the instrument performance we have been assessing it by distributing to the labs hard dye beads (like CS&T beads from BD and rainbow 8 peak beads) and lyophilized pre-stained compensation beads (this compensation beads are labeled with each of the fluorochromes that are present in the panels, ie FITC, PE, etc).
As you can see in this table we are able to calculated different parameters that are related to instrument performance:
With the compensation beads we calculate the Stain Index, a measure of resolution. It takes into account how separate are the + and neg peaks, and how much spread there is in the negative population. A higher stain index means higher resolution. As you can see the SI varies across labs, numbers in red indicating the lower ones.
With the CS&T beads we calculated Qr values that are an indication of the efficiency for each detector. A high Q values translates in high resolution.
Also with the CS&T beads we can calculate a value called Br that is a reflection of background for each detector. A low B value translates in high resolution.
We also looked at the pattern of 8 peak beads.
As you can see for example lab 2 has a high stain index and can resolve the 8 peak beads. This very good resolution correlates with a high Q value and a low B.
In contrast labs 6 and 7 have low stain index, in the 8 peak beads the peaks at the low end are not resolved and that correlates with low Q values (specially lab 6) and high Br values.
Maybe this slide will also benefit from some sort of animation?
Low SI = Bad Resolution;
correlates with low Qr and high Br
Low SI = Bad Resolution;
correlates with high Br
Impact of Instrument Performance in Quality Assurance of Multicolor Flow Cytometry Assays.
Jaimes MC,1 Stall A,1 Inokuma M,1 Hanley MB,1 Maino S,1 D’Souza MP,2 and Yan M.1 ISAC 2010
1BD Biosciences, San Jose, CA 95131; 2Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 62

63. Correlation of SI, Qr, and Br with Assay Performance
Lab 2
Lab 4
Lab 6
Lab 7
Plots gated on lymphocytes
Plots gated on CD3+ cells
How this information on instrument performance translates in assay performance?
Here is the data from some of the labs discussed in the previous slide.
If you look on the first row at CD3 resolution, you will see that for some labs like lab 7, the distance between + (blue population) and negative (red population) is small compared to other labs (like 6 or 4) meaning that there is a difference on the resolution for this channel. Despite this, in this example all labs will be able to gate in the population of interest.
However if you look to the plots in the middle you can see how if the resolution of some population is poor this has an impact on the data. In this case lab 6 has very low resolution for CD8(y axis, green and purple populations are very close compared to other labs) and CD4 (x axis, green and blue population). These problems are shown by the black arrows.
Importantly the difficulty at gating on CD8 or CD4 will have an impact on the ability to detect cytokine responses as seen in the data in the bottom row.
Plots gated on CD3+CD8+ cells
Impact of Instrument Performance in Quality Assurance of Multicolor Flow Cytometry Assays.
Jaimes MC, Stall A, Inokuma M, et al. ISAC 2010 63

64. 1 Assay – 4 Platforms CD4 V450 CD8 PE CD3 FITC CD20 APC
BD FACSCanto II
BD LSR II
BD FACSAria III
BD LSRFortessa

65. Thank You!!

66. Defining Instrument Performance and Sensitivity: Qr, Br, and SDen

67. Qr: Anti-CD10 PE Example Dim Population
Trans-
mission SI
Corrected
The laser and detectors were attenuated by ND filters over a 30-fold range to illustrate the effects of decreasing detector sensitivity on population resolution.
Qr=0.227
100% 83
Qr=0.087
35.5% 55
Qr=0.038
17.8% 35
Qr=0.014
7.1% 20
Qr=0.007
3.5%

68. Relative Background: Br
Br is a measure of true optical background in the fluorescence detector, which helps indicate how easily (dim) signals may be resolved from unstained cells in that detector.
Scatter from the flow cell and ambient light.
Unbound antibody or fluorochrome
Raman scatter
Spectral overlap on a cell
Cell autofluorescence
It measures the variation (CV) of the noise due to optical background.
Increased background light broadens the distribution of unstained or dim particles and results in decreased resolution sensitivity.
Raman scatter is a scattering of light due to water molecules found in sheath fluid or sample buffer. Raman scattering is highest in the PE parameter for blue laser excitation.
Cell autofluorescence is a type of fluorescence that’s inherent to the cell itself being excited by the laser.
Factors affecting Br: dirty flow cell, damaged optical component

69. Why is Br important? High Br widens negative and dim populations.
High Qr value = lower resolution
Low Qr value = higher resolution
Low Br
High Br

70. Br: Optical Background from Propidium Iodide
Example: It is common to use propidium iodide (PI) to distinguish live from dead cells. Propidium iodide was added in increasing amounts to the buffer containing beads, and Qr and Br were estimated.
PerCP
1000
2000
3000
4000
5000
6000
7000
8000
9000 1 2 3 4 5 6
PI-free dye (µg) Br
0.01
0.02
0.03
0.04
0.05 Qr
Residual PI in your sample tube will increase Br, which will reduce sensitivity.

71. Electronic Noise (SDen)
SDen is the background signal due to electronics:
Contributed by:
PMT connections / PMT noise
Cables too near power sources
Digital error
Broadens the distribution of unstained or dim particles
Increases in electronic noise results in decreased resolution sensitivity
Most important for channels with low cellular autofluorescence
APC-Cy™7, PE-Cy™7, PerCP-Cy™5.5
Cy™ is a trademark of Amersham Biosciences Corp. Cy™ dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ , USA.

72. Gel (from coupling) found all over the flow cell????
Tiff file missing

73. Contributions to Background Br
Br is a measure of true optical background in the fluorescence detector, which helps indicate how easily (dim) signals may be resolved from unstained cells in that detector.
Scatter from the flow cell and ambient light.
Unbound antibody or fluorochrome
Raman scatter
Spectral overlap on a cell
Cell autofluorescence
It measures the variation (CV) of the noise due to optical background.
Increased background light broadens the distribution of unstained or dim particles and results in decreased resolution sensitivity.
Raman scatter is a scattering of light due to water molecules found in sheath fluid or sample buffer. Raman scattering is highest in the PE parameter for blue laser excitation.
Cell autofluorescence is a type of fluorescence that’s inherent to the cell itself being excited by the laser.
Factors affecting Br: dirty flow cell, damaged optical component

78. Grab this slide from the CS&T PPT

79. Get the animated slide from multi-color or CS&T PPT