Designing and Implementing a High-Level Multicolor Flow Cytometry Assay. Brent Wood MD PhD Department of Laboratory Medicine University of Washington

Transcription

1 Designing and Implementing a High-Level Multicolor Flow Cytometry Assay Brent Wood MD PhD Department of Laboratory Medicine University of Washington

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

3 Outline Instrument optimization Compensation Reagent selection and optimization Panel validation Data analysis

4 Detector Optimization

5 Detector Optimization

6 PMT Voltage Green = 400 V, Blue = 450 V, Red = 500 V, Violet = 550 V

7 Detector Voltage 475 volts 575 volts 675 volts 775 volts 875 volts Too low reduces sensitivity Too high pushes bright signals off scale Dilute with unlabeled antibody May need to compromise

8 Optimization Daily optimization not practical Once optimized, do NOT change instrument settings unless change in performance New optical filters New PMT detector Monitor performance daily Use stable bright beads Assess MFI and CV for each detector Levy-Jennings plots Define acceptable ranges of performance

9 Fluorochrome Intensity CD4 FITC = Green PE = Dark Blue PE-TR = Light blue PerCP-Cy5.5 = Magenta PE-Cy7 = Orange Pacific Blue = Red A594 = Yellow green APC = Light green A700 = Blue APC-Cy7 = Purple

10 Tandem Fluorochromes Phycoerythrin (PE) PE-Texas Red

11 Tandem Breakdown Whole blood lysis Cytometry Part A (2009) 75A: NH 4 Cl prelyse and wash 1 hour prior

12 Fluorochrome Options PE-Cy5 vs. PerCP-Cy5.5 vs. PE-Cy5.5 vs. PerCP PerCP dim PerCP-Cy5.5 brighter but dims with increased laser power PE-Cy5 brighter but significant comp with APC PE-Cy5.5 bright, less comp with APC, more comp with PE-Cy7 and APC-A700 A700 vs. APC-A700 APC-A700 significantly brighter but more comp with APC APC-Cy7 vs. APC-H7 vs. APC-A750 APC-Cy7 undergoes photodegradation but is brightest APC-H7 dimmer but more stable, low comp with APC APC-A750 intensity in between but higher comp with APC

13 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

14 FITC = Green PE = Orange Excitation = Dotted Emission = Solid

15 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

16 Compensation Correct Undercompensated Overcompensated

17 Compensation

18 Compensation

19 Compensation Don t worry unduly about PMT voltage 245% 103% 47.5% 23.0% 12.2% 1.9% 4.7% 10.3% 21.0% 41.0% PE = 400 volts PE-TR = 550 volts 450 volts 500 volts 550 volts 600 volts Compensation values should reflect relative spectral overlap, i.e. detector gains should be equal

20 Compensation Validation Fluorescence minus one FMO controls

21 Compensation Validation Fluorescence minus one FMO controls

22 Compensation Quality Control Purist Set at the time of each experiment Important when: Reagents and specimen preparation are inconsistent Accurate MFI to be measured Practical Quality control instrument and reagents using consistent specimen preparation Compensation is dependent on relative fluorescence intensity Slight manual adjustment may be required

23 0.9% 0.9% 1.0% June 2008 Nov 2008 Sept % 1.0% 1.1% May 2008 Dec 2008 May % 4.5% 6.1% May 2008 Dec 2008 May 2009

24 Stem cell evaluation Antigens to be evaluated: Early progenitors - CD34, CD117, CD38 Erythroid - CD71 B cell - CD19 pdcs, basophils - CD123, HLA-DR Myelomonocytic - CD15, HLA-DR Progenitor gating - CD45 Test reagents - CD45RA, CD133, CD7, etc.

25 Surface Antigen Titration 2 ul + 5 ul + 10 ul Titer for signal to noise Saturation desirable Use same total volume as assay Use 5 ul

26 Cytoplasmic Antigen Titration 10 ul, 5 ul, 2 ul, 1ul of neat Titer for signal to noise Not saturation Particularly important for cytoplasmic antigens 10 ul, 5 ul, 2 ul, 1ul of 1:10 Use 5 ul of 1:10 Use 10 ul of 1:10

27 Possibilities PB / V450 DR CD15 CD19 CD123 CD117 CD38 CD34 CD71 CD45 X FITC PE PE-TR P-X PE- Cy7 A594 APC APC- A700 APC- X7

28 Possibilities PB / V450 DR CD15 CD19 CD123 CD117 CD38 CD34 CD71 CD45 X FITC PE PE-TR P-X PE- Cy7 A594 APC APC- A700 APC- X7

29 Fluorochrome Matching Match level of expression with fluorochrome intensity Bright expression = Dim fluorochrome Dim expression = Bright fluorochrome

30 CD34

31 CD123

32 Background Autofluorescence varies between populations Fluorochrome may give non-specific binding Cyanine containing PE tandems Monocytes Fc-receptor mediated Beckman-Coulter reagents block PE-Cy5 PE-Cy5.5 PE-Cy7 Idiosyncratic CD30 APC-A700 Important gating reagents should be clean

33 Possibilities PB / V450 DR CD15 CD19 CD123 CD117 CD38 CD34 CD71 CD45 X FITC PE PE-TR P-X PE- Cy7 A594 APC APC- A700 APC- X7

34 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

35 Adjacent fluorochromes

36 Primary of Tandems 10.5% 1.6%

37 Interlaser compensation

38 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

39 Avoid bright fluorescence

40 Different populations

41 Bright dual positive

42 Possibilities PB / V450 DR CD15 CD19 CD123 CD117 CD38 CD34 CD71 CD45 X FITC PE PE-TR P-X PE- Cy7 A594 APC APC- A700 APC- X7

43 Possibilities PB / V450 DR CD15 CD19 CD123 CD117 CD38 CD34 CD71 CD45 X FITC PE PE-TR PECy5 PE- Cy7 A594 APC APC- A700 APC- X7

44 Possibilities PB / V450 DR CD15 CD19 CD123 CD117 CD38 CD34 CD71 CD45 X FITC PE PE-TR PECy5 PE- Cy7 A594 APC APC- A700 APC- X7

45 Final PB / V450 DR CD15 CD19 CD123 CD117 CD38 CD34 CD71 CD45 X FITC PE PE-TR PECy5 PE- Cy7 A594 APC APC- A700 APC- X7

46 Compensation Compromises

47 Validation Fluorescence minus one controls Antibody interactions Avoid IgG2 class unless plasma removed Understand background Troubleshooting Run many samples Under same conditions as to be used Positive and negative controls

48 PE background

49 Data analysis Need better software tools Multiparametric analysis is essential Population identification Compensation background Informative use of color Density plots very helpful Use many dot plots Not every possible combination

50 Erythroid gating

51 Information Opportunities

52 Information Opportunities

53 Information Opportunities

54 Conclusion Instrument optimization Reagent selection Fluorochrome matching Fluorochrome options Background evaluation Compensation background Develop compensation strategy Validation Develop data analysis strategy