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

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Presentation on theme: "Issues in Multicolor Flow Cytometry: Beyond 6 Colors Brent Wood MD PhD Department of Laboratory Medicine University of Washington."— Presentation transcript:

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 Insensitive Misattribution if assumptions incorrect % 80% 66% 0.4%

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 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 N = 35

11 Euroflow

12 Panel Design PBFITCPEPE-TRPE55PE7A594APCA700APC7 B cells 45  T cells Blasts DR Myeloid DR

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

17

18 0.1% abnormal immature B cells ALL MRD

19 Tandem Fluorochromes Phycoerythrin (PE)PE-Texas Red

20 Tandem Breakdown Whole blood lysis NH 4 Cl prelyse and wash 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 CorrectUndercompensatedOvercompensated

25 Compensation Don’t worry unduly about PMT voltage 245% 1.9% 103% 4.7% 47.5% 10.3% 23.0% 21.0% 12.2% 41.0% 500 volts550 volts 600 volts450 voltsPE = 400 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

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 Bendall, et al (2011) Science 332: parameters

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

45 Conclusion Multicolor flow 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


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