1. Issues in Multicolor Flow Cytometry: Beyond 6 Colors
Brent Wood MD PhD
Department of Laboratory Medicine
University of Washington

2. The Power of Flow Cytometry
Single cell analysis
Multiparametric
Rapid
Quantitative
Flexible

3. Inferential reasoning
18%
80%
66%
0.4%
Insensitive
Misattribution if assumptions incorrect

4. Analytical Approach Antigen expression is present/absent
Antigen intensity is minimally used
Can be expressed as a table of percentages
Focus is on abnormal immunophenotype
Presence or absence of expression of antigens

5. Direct Observation
Combination of reagents uniquely identifies cell type, lineage and maturational stage
Emphasize normal maturational patterns
Direct determination of immunophenotype without inference
Improvement in sensitivity and specificity
More simultaneous fluorochromes improves

6. Multiparametric Flow Cytometry
More accurate population identification
Greater informational content
Make better use of small specimens
Fewer cells, more information
Process fewer tubes
Save on reagents, tech and instrument time
Collect large number of events efficiently
Allow standardized reagent combinations

7. Instrumentation Beckman-Coulter FC500 Becton-Dickinson FACSCanto
5 colors - 1 or 2 lasers
Becton-Dickinson FACSCanto
I - 6 colors, 2 lasers
II - 8 colors, 3 lasers
Beckman-Coulter Gallios
10 colors - 3 lasers
Becton-Dickinson LSRII
~20 color, up to 7 lasers

8. How Many Colors are Enough?
Ideal
Add all reagents of interest into single tube
Real
Too many parameters of interest

9. Define Purpose of Assay
Most important question
What information is required?
What information is most important?
Prioritize
Compromises are inevitable
Simplest assay is best

10. Bethesda International Consensus Conference

11. Euroflow

12. Panel Design PB
FITC PE
PE-TR
PE55
PE7
A594
APC
A700
APC7
B cells 45 k l 19 34 20 38 10 - 5
T cells 2 7 8 3 4 56
Blasts DR 15 33
117 13 71
Myeloid 64
123 14 16

13. Cell Type Identification
Borowitz et al (1993) AJCP 100:
Steltzer et al (1993) Ann NY Acad Sci 667:

14. Normal Blast Maturation
Wood (2004) Methods Cell Biology 75:

15. Acute Myelomonocytic Leukemia
Wood and Borowitz (2006) Henry’s Laboratory Medicine

16. Hodgkin Lymphoma

18. ALL MRD
0.1% abnormal immature B cells

19. Tandem Fluorochromes
Phycoerythrin (PE)
PE-Texas Red

20. Tandem Breakdown NH4Cl prelyse and wash Whole blood lysis 1 hour prior
Cytometry Part A (2009) 75A:

21. Compensation Spectral overlap between fluorochromes
Critical to success of method
For 10 color experiment
Need to determine 90 values for Comp Matrix
Software compensation required
Maximum flexibility
Non-destructive

22. FITC = Green
PE = Orange
Excitation = Dotted
Emission = Solid

23. Compensation - Method Single stained controls used
One for each individual fluorochrome
One for each individual tandem
As bright as brightest reagent to be used
Samples run without compensation
Compensation calculated in software
Applied either at acquisition or analysis

24. Compensation
Correct
Undercompensated
Overcompensated

25. Compensation Don’t worry unduly about PMT voltage PE = 400 volts
245%
103%
47.5%
23.0%
12.2%
1.9%
4.7%
10.3%
21.0%
41.0%
PE = 400 volts
450 volts
500 volts
550 volts
600 volts
PE-TR = 550 volts
Compensation values should reflect relative spectral overlap, i.e. detector gains should be equal

26. Compensation Validation
Fluorescence minus one “FMO” controls

27. Compensation Validation
Fluorescence minus one “FMO” controls

28. Compensation

29. Compensation

30. Compensation Background
Avoid increased background due to fluorochromes
Adjacent with longer wavelength emission
PE / PE-TR, PE-TR / PE-Cy5, PE-Cy5.5 or PerCP-Cy5.5/ PE-Cy7
APC / APC-A700, APC-A700 / APC-Cy7
Primary fluorochrome of tandem
PE and PE-TR, PE-Cy5, PE-Cy5.5, or PE-Cy7
APC and APC-A700 or APC-Cy7
Interlaser excitation and emission
PE-Cy5 and APC
PE-Cy5.5 or PerCP-Cy5.5 and APC-A700
PE-Cy7 and APC-Cy7
PE-TR and A594

31. Adjacent fluorochromes

32. Primary of Tandems
10.5%
1.6%

33. Interlaser compensation

34. Strategies to deal with compensation background
Avoid bright fluorescence
Put fluorochromes on different populations
Put fluorochromes brightly on same population
Avoid detection of dim expression in presence of high background

35. Avoid bright fluorescence

36. Different populations

37. Bright dual positive

38. Compensation Compromises

39. Infinicyte - Cytognos
Nearest neighbor estimate of relationship between parameters in different tubes
Pedreira, et al. Cytometry (2008) 73A:

40. Mass Cytometry
simultaneous antigens

41. Mass Cytometry
Bendall, et al (2011) Science 332:687

42. Mass Cytometry
Bendall, et al (2011) Science 332:687

43. Mass Cytometry
13 parameters
Bendall, et al (2011) Science 332:687

44. Mass Cytometry 18 functional markers 13 conditions
Bendall, et al (2011) Science 332:687

45. Conclusion Multicolor flow cytometry Mass Cytometry Powerful tool
Purpose must be primary consideration
Simpler is better
Fluorescent spectral overlap is major limitation
Mass Cytometry
Allows high level multiparameter measurements
Eliminates fluorescent spectral overlap
Detection sensitivity and reagent availability are concerns
Both require improved data analysis tools