Visualization of digital data Interpretation of the visual

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1 Technical aspects of 8-color flow cytometry in the diagnosis and classification of hematopoietic malignancies Tomas Kalina!"#$%&'()*+,&$'+-./(0 *1 (2#34%-.(56(7&1+3+*&/( 8$#94&/(!:&3"(;&<4=%+3( Discovery or Artifacts? CD3+ lymphocytes Uncompensated, single stain CD3+ lymphocytes, correctly compensated C(!"+%1"551(D&4E&A+#(F*,&'G9#G5*(8$#94&( Compensation skewing Visualization of digital data Other names: Log-linear transformation Logicle display Biexponential (DiVa) VisiComp (Summit 4,3) Herzenberg, L. A., J. Tung, et al. (2006). "Interpreting flow cytometry data: a guide for the perplexed." Nat Immunol Visualization of digital data Interpretation of the visual 1

2 Technical basics PMT settings (CS&T module) Compensation settings (comp wizards) Fluorochrome choice Panel design Inter laboratory comparisons Interpretation of the data: Are the data technically OK? Background? Sensitivity? What is dim/negative? Dim/bright? How was the gating done? How to get the same data in a multi-center project? What are the best fluorochromes? What are the best sample preparation protocols? How to make it work all together? Target fluorescence channels for the 8 th Rainbow bead peak (lot: X02) H7-A: CD8 LOGICAL El. noise( Target values (lot: X02 of 8 th Rainbow bead peak) Target MFI PB PO PE Cy7-A: CD16 LOGICAL Reference value PE No bright positive population out of the window of analysis - Reference values in the plateau part of the curve in all instruments PE Cy H Ref. :Maecker, H. T. and J. Trotter (2006). Cytometry A. 2

3 Initial selection Further comparisons Relative brightness Excitation & emission profile Type of sample/cells Instrument optical configuration PE Cy7 H7 Cy7 PE TxRed PE Spillover into other channels Panel aim Availability 1 Violet 2 Violet 3 Blue Commonly available fluorochromes 5 Blue PE-TxRed 6 Blue 8 Red 9 Red Cy7 H7 1 Violet 2 Violet 3 Blue Commonly available fluorochromes 5 Blue PE-TxRed 6 Blue 8 Red 9 Red Cy7 H7 Intensity fluo: 8-colors Spillover fluo: Fluorochromes 1 Violet 2 Violet 3 Blue 6 Blue 8 Red 9 Red H7 Single fluorochrome dyes PE Generic reagent independent compensation Tandem dyes PE Cy7 H7 Reagent conjugate & lot specific needs Generic and reagent/lot-especific Compensation tubes Generic PE-Cy7 conjugates -H7 conjugates CD20 PB CD2 CD3 CD45 PO CD8 CD4 CD8 CD10 CD8 CD8 PE CD16 CD9 CD5 Cy5-5* CD19 CD10 CD8 CD45RA CD14 CD45RO CD19 CD56 CD24 CD117 CD38 HLADR CD43 *Tandem dye CD49d CD71 CD81 Lambda Design the compete panel first Match the single stained compensation tubes to the panel The same negative and positive reference populations Beads x Beads OR Cells x Cells 3

4 GOALS: * Adequate scatter resolution of major cell populations * Minimal cell loss * Preservation of fluorochrome brightness * Low impact on stability of fluorochrome tandems * Easy and fast to use * Reduced data acquisition time Titration of reagents: Titrate for final volume+time Reach the saturation conditions Watch the background on negatives 10 µl 7 µl 5 µl 2,5 µl 1,25 µl 0,625 µl 0 µl What happens if AmCy channel moves 10%? What happens if AmCy channel moves 10%? to spill, compensated -10 % MFI value, compensated -10 % MFI value, compensated Exact MFI value, compensated Exact MFI value, compensated +10 % MFI value, compensated +10 % MFI value, compensated Euroflow standard procedure: Erythrocyte lysis, time to acquisition, fixation? Preservation of scatters, cell loss, fluorochrome brightness and stability, ease of use, reproducibility Analysis 4

5 10/2/10 Summary 8 centers 1 stabilized blood sent around 3-4 healthy donors recruited at each center Software data merging and analysis Reproducible and standardized data CD20 CD45 CD8 CD27 CD4 CD19 CD14 CD3 Well founded consensus - > accepted and useful SOP Well thought settings and regular quality control Tested, reviewed reagents, fluorochromes, procedures 5