Understanding Flow Cytometry

Transcription

1 Understanding Flow Cytometry The Basic Concepts Maree Bagnara Products Sales Specialist/Account Manager Flow Cytometry PN Successful Flow Cytometry data is driven by.. Understanding the Biology Basic Immunology Population(s) of interest Antigen Expression Dim/bright Ag proximity Intracellular/extracellular Understanding the Technology Fluorochrome Excitation / Emission spectra Instrument Platform Lasers, Colour capability, Filters Spectral Overlap - Compensation Reagent and Assay Optimization Understanding of application methods GOOD SAMPLE PREPARATION 2 1

2 Basic Immunology Fluorescence Techniques Direct Staining Moab against target species Directly tagged with Fluorochrome Multi colour staining Indirect Staining Mouse anti human CD4 Human CD4 Goat anti-mouse FITC Amplification of low density Ags 3 Typically single colour 4 2

3 Fluorochrome Options for Gallios QDots, Hoechst PI, 7AAD, PerCP, GFPs Exercise 2 5 Excitation/Emission of Common Fluorochromes 525nm 575nm 620nm 675nm 700nm 770nm FITC PE ECD PC5 PECy5.5 PC7 488nm 647nm 675nm 700nm 700nm 750nm 750nm APC Alexa 700 APCAlexa 700 APC-H7 APCAlexa nm Pacific Blue 450nm Pacific Krome Orange Orange Sensitive to light 6 3

4 Measuring Brightness Quantitation of Brightness over Background Current method Signal:Noise MFI(pos) / MFI(neg), Alternative Method Stain Index (Sensitivity) MFI(pos) MFI(neg)/ 2 x SD Background Takes into account the spread of the background (negative population) vs positive population More accurate S:N References Maecker et.al Cytometry 62A: (2004) Hulspas et.al Cytometry 76B : (2009) Measuring Brightness Quantitation of Brightness over Background Signal to Noise Ratio: MFI(pos) / MFI(neg), /0.37 = 1136 Staining Index: (MFI(pos) MFI(neg))/2*SD(neg), ( )/2*0.23 = 913 Information provided courtesy of Dr Michael Kapinsky, Beckman Coulter 4

5 Compensation 9 Compensation Exercise

6 Tandem Dye Technology Schematic of Energy Transfer Mechanism 488 nm LASER Donor Molecule Excitation PE Acceptor Molecule Emitted wavelength Cy5 Donor Molecule Acceptor Molecule PE (488) Texas Red PE (488) Cy5 PE (488) Cy7 PerCP (488) Cy5.5 APC (635) Alexa700 Commercial Product ECD PC5 PC7 PerCPCy nm APC Alexa nm 630 nm 675 nm 760 nm 700nm 11 Tandem Dye Technology Better sensitivity over synthetic dyes Not equivalent from manufacturer to manufacturer Issues Variation in energy transfer Impacts compensation/instrument setup Photostability Stability Binding Specificity Cy5 can bind nonspecifically to monocytes Lot to lot variability 12 6

7 Same Tandem Different Vendor 13 Conjugate Choices High density Ag/ Dim dye Fluorochrome choice dependent on antigen density Bright dye - low density antigens Dimmer dye - high density antigens Low density Ag/ Bright dye Critical for multi-color reagents Proper compensation Detection of dim/rare events Dye conjugate interactions do they work together? Non-specific binding Steric Hindrance Fluorescent quenching 14 7

8 PBPs, Tandems, or Synthetic Dyes? Intracellular Antigen Detection Cytoplasmic antigens: Phycobiliproteins (PE, APC), Synthetic Dyes (Cyan and Alexa Dyes)best Tandem dyes can be degraded by cellular enzymes Alexa Fluor 488 better than FITC lower background Nuclear antigens: Phycobiliproteins or tandem dyes: protein size may hinder binding Close proximity can lead to energy transfer between dyes 15 Compensation Normal Human Lymphocytes 16 8

9 Compensation Normal Human Lymphocytes 17 Understanding Colour Compensation Back to Basics Set-up for Compensation Neg controls to set PMT HV Isotype matched and /or blank sample Place negative cells in the first decade Single positive controls for each fluorochrome / CD8 of each flurochrom Use biological sample or antibody capture beads such as VERSACOMP Verifier tube to check settings Set up a single protocol with histograms for all possible fluorochrome combinations Run each tube through protocol, making adjustments to compensation as required Final tube should be a verifier which checks that the settings are correct 18 9

10 19 Compensation Matrix 20 10

11 Compensation Adjustment 21 Spillover Guide for the Gallios U N T O U C H A B L E Untouchable = No overspill from other dyes (clean row) U N T O U C H A B L E S I L E N T Silent = No overspill into other channels (clean column) Classification is specific for each combination of antibodies and conjugated dyes on a given hardware configuration Information provided courtesy of Dr Michael Kapinsky, Beckman Coulter 11

12 Multi-Color Combinations: What Can Go Wrong? Charge Size Proximity Concentration Conjugate interactions can make the difference between right and wrong results Decreased Mean Fluorescence or decreased % POS 23 Non-Specific Binding of Dyes Root Cause Low affinity binding of conjugate to irrelevant populations usually FcR Specific to cyanine dyes Other large molecule conjugates also display problem Monocyte binding of Cyanine dye Blocking antibody - unlabelled Same species as test Ab Bind to FcR Use Ig fraction rather than serum 30ug Ig fraction per 10 6 cells in 100 ul

13 Optimising Moabs Perform titration curves for each conjugate by serial dilution assay Plot Fluorescence Intensity vs dose for both positive and negative signal Choose optimal dose Saturation area of curve Highest Signal to Noise Prepare combination for Moab Cocktails Matrix Titre Evaluate for Non-specific binding Steric Hindrance Fluorescence Quenching 25 Dose Titration Dilution Perform titration curves for each conjugate by serial dilution assay Mean Channel Mean Channel MoAb conjugate positive pop negative pop 1:8 1:4 1:2 NEAT Plot Fluorescence intensity vs dose for both positive signal and noise Determine Signal/Noise Ratio Choose optimal conc range (2-3 points) Saturation area of curve Highest S/N Ab conc./test (μg) / Dilution 26 13

14 Moab Cocktail Optimisation Use titration information for Moab cocktail matrix CDx at three doses 1,2,3 CDy at three doses a,b,c Ideal [Ab] ug/test staining 1X10 6 cells in 100ul Determine saturation point Note: each Moab volume dilutes the other! Evaluate performance for major interactions Non-specific binding Steric Hindrance Fluorescence Quenching Verify on multiple samples x N 1:2 1:4 N 1:2 1:4 y 27 Optimal Formulation 20.0 Single CD4 FITC 20.0 CD4 FITC (with CD8 PE) Fluorescencence S/N Positive MFI Negative MFI Fluorescencence S/N Positive MFI Negative MFI Dose (ug/test) Dose (ug/test) Formulation Choice Choose highest Signal/Noise within saturation area of curve Moab mix must demonstrate equivalent performance to single color reagent controls No evidence of any interactions 28 14

15 Specimen Processing Sample presentation Anticoagulants ETDA, Heparin Cell Lines Adherent, Non-adherent Cell Media Phenol Red Clumping Flow Cytometer nightmare! Age of sample EDTA 8 30 hours Gate out dead cells Dye for Viability PI, 7AAD Red Cell Contamination Many RBC Lysis Products Optilyse Immunoprep Ammonium Chloride Ficoll Hyapaque 29 Sample Preparation Pre and Post Wash Remove interfering dyes from Culture medium Post wash can improve signal to noise Loss of Cells Washing Medium Check ph of PBS ph7.2 FCS? B etter than BSA? Check ph after adding buffering serum Autofluorescence Gate autofluorescence population and backgate onto Scatter plot ensure cells of interest are not contained 30 15

16 Sample Preparation Antibody-Antigen binding is a dynamic process Influenced by three parameters Time 1min 15mins Size of fluorochrome, cocktail size, Ag density Temperature Room Temperature. If delay - refrigerate Concentration of Moab Use in excess rather than less Are you using adequate Moab for the worst" case scenario Questionable data?» Use it at manufacturer s recommendation on a normal specimen.» Run Moabs as a SINGLE stain if using in a cocktail 31 Inhouse Cocktails! Do not store pre-mixes for extended periods of time Do NOT dilute / pre-mix Moab in original vial Do a time course study Add protein Dark vials When in doubt run a normal control specimen! 32 16

17 Negative Controls what do they do? Fluorescence due to non-specific binding of MoAb to cell Mainly due to binding to Fc receptors on the cell surface Dependant on IgG subclass IgG1<IgG2a~IgG2b<IgM (Most sticky ) 33 Why Use Negative Controls Set fluorescence PMT HV to obtain best signal to noise ratio Usually in the first decade Set Analysis regions 0.1-2% 34 17

18 Negative Control A non-specific MoAb of the same IgG subclass as the specific MoAb used to assess positive staining Should be at the same protein concentration as the MoAb Should have the same Fluorchrome/Protein ratio as the specific MoAb More common to use the cells that stain negative for the moab of interest internal Control 35 Where do we put the region? Negative 2% Positive R 1 Often needs to be moved with test samples 45% R 1 Subjective. Move the region if necessary! 38% R 1 36 Fluorescence 18

19 Where do we put the region? 37 Low Ag density on cells How do we handle this sample? 30% pos or 100% weak pos? Cells that are negative for an Ag may be more useful than a neg control Negative control Specific stain R1 (Log) Fluorescence 38 19

20 Application Standardisation Standardise the fluorescent light emitting from the Photo Multiplier Tubes Enables data comparison between samples and across instruments Maintain compensation settings 39 Mean Channel Standardisation-Using FlowSet beads PMT HV = 456 V PMT HV = 456 V DAY 24 DAY 0 DAY 24 - Drug XYZ DAY 24 HV to 350 V Decrease PMT HV to 350 V 40 20

21 Standards In Cytometry 41 21