IV. International Symposium Flow Cytometry/Molecular Biology HOSPITAL ISRAELITA ALBERT EINSTEIN São Paulo 2012 Quality Control in polychromatic Flow Cytometry - EuroFlow interlaboratory standardization Tomáš Kalina Charles University, 2 nd Faculty of Medicine, Prague, Czech Republic Dpt. of Pediatric Hematology and Oncology - Childhood Leukemia Investigation Prague
Flow cytometry in clinical decision making Flow Cytometry in hematology: Malignant vs. Reactive Leukemia phenotype - lineage or more? Minimal residual disease How sensitive? How reliable? False positives/false negatives Robustness -> Interpretation -> Understanding
Leukemia phenotype - lineage ALOT (Acute Leukemia Orientation Tube) n=152 dg cases, 8 EuroFlow labs BCP-ALL cases (n=89) T-ALL cases (n=27) AML cases (n=36) slide designed by L. Lhermitte, ALL panel coordinator
Flow cytometry data interpretation FCS data: Pattern analysis Errors is it for real? Comparison to reference Can you interpret somebody else s FCS data? What is limiting inter-laboratory collaboration? Variability (pre-analytical and analytical) day-by-day, instrument-to-instrument lab-to-lab
Limiting variability - standardization
What is the key feature of FCS data? Patterns Fluorescence intensity Relative fluorescence Factors setting the fluo intensity: PMT settings Reagent (clone, fluorochrome) Compensation (artefacts?) Sample preparation
8-color flow cytometry of hematological malignancies www.euroflow.org
Standardization and QC Median fluorescence intensity of the bright peak CV of the bright peak Daily QC with Rainbow x CS&T beads by BDB
EuroFlow target values Lot: Lower MFI (-15% ) Target MFI Rainbow 7-peak (DiVa) Upper MFI (+ 15% ) EAB01 Pacific Blue 100,452 118,178 135,905 Pacific Orange 93,871 110,436 127,002 FITC 28,752 33,826 38,900 PE 32,381 38,095 43,809 PerCP 66,846 78,642 90,438 PC7 8,316 9,783 11,250 APC 158,639 186,634 214,629 APC-H7 64,194 75,522 86,850 Rainbow 8-peak beads (Spherotech) EuroFlow lot EAB01, EAC01
Standardized data - EuroFlow Kalina, Leukemia, 2012
How to keep control of standardized data? QC rounds: The old way (ISHAGE, UK NEQUAS) report percent positive of given subset standard (fixed) sample sent around EuroFlow QC report fluorescence intensity standard instrument setup, sample prep, gating no sent around sample Euroflow QC setup Single 8-color tube ( LST QC tube ) > stain 3 donors Acquire 3 tubes + Rainbow beads Gate using defined gating -> report to table Upload all FCS files + filled table
LST-QC tube (Lymphocytosis screening tube) The limited LST (8 color / 11 markers) CD20+CD4 PacB / CD45 PacO / CD8+Lambda FITC /CD56+Kappa PE / CD5 PerCP55 /CD19 PC7 / CD3 APC/CD81 APC-H7 10 EuroFlow laboratories 12 instruments (11x BD Canto II / 1x BD LSR II) 3 healthy donors in each lab 3 rounds (once a year)
CD19 CD8 CD19 CD5 CD19 Lambda LST-QC tube - gating B-cells / Kappa vs. Lambda T-cells / CD4+ vs. CD8+ CD3 Kappa NK-cells CD3 CD4 CD3 The limited LST (8 color / 11 markers) CD20+CD4 PacB / CD45 PacO / CD8+Lambda FITC /CD56+Kappa PE /CD5 PerCP55 /CD19 PC7 / CD3 APC/CD81 APC-H7 CD56
Cross-platform gating is it possible? Can we merge and analyze together data from different flow cytometers by different manufacturers?
Cross-platform comparison Instrument Resolution Manufacturer Fluor chnl. Canto II Cyan ADP Navios MACS Quant 18bit/26214 4 12bit/ 4096 20bit/10485 76 405nm 488nm 633nm BDB 8 color 2 / 4 / 2 Dako/BC 9 color 2 / 5 / 2 Beckman- Coulter 10 color 2 / 5 / 3 Miltenyi 8 color 2 / 4 / 2 Common Fluors PacB-PacOr / FITC-PE-PerCPCy55-PC7 / APC- APCH7
Cross-platform comparison - Target values How to get the values for non DiVa based cytometers: MACS Quant simple recalculation Cyan ADP export Rainbow FCS 2.0 from DiVa, import to Summit, read values Navios measure Rainbow at increments of 50mV, rescale to 18bit, regression analysis to obtain PMT values
Unstained accross all platforms Navios MACSQuant Cyan ADP Canto II Histogram with medians 4 platforms / 4 files
Immunophenotype accros all platforms B-cell Kappa+ T-cell CD4+ B-cell Lambda+ T-cell CD8+ NK cells APS view 4 platforms / 12 files
Summary Standardization ensures comparability of the flow cytometry data : day-by-day, instrument-to-instrument lab-to-lab Fluorescence intensity is the primary parameter we use in flow QC based on MFI of gated subsets is feasible Coefficient of Variability in 3 years EuroFlow trial below 60% (below 30% in half of the parameters) Expression levels of the selected markers on human lymphocytes are well conserved -> no need for fixed sample sending Different flow cytometry platforms from diverse manufacturers can be used Standardized cell subsets fluorescence allows for easy and reliable clustering in multicolor space QC is capable to show the irregularities, problems -> EuroFlow is developing a QC testing scheme for the community of the EuroFlow approach users
Thanks! Michaela Nováková Marcela Vlková - Childhood Leukemia Investigation Prague Juan Flores Monterro Quentin Lecrevisse
EuroFlow 2006 2012..and going on