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Data Analysis . Department of Laboratory Medicine University of Washington. Data Analysis. Assess data quality Remove artifacts Identify populations Compare with normal Identify abnormal populations Quantitate and evaluate immunophenotype Generate report. Assess Data Quality.
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Data Analysis Department of Laboratory Medicine University of Washington
Data Analysis • Assess data quality • Remove artifacts • Identify populations • Compare with normal • Identify abnormal populations • Quantitate and evaluate immunophenotype • Generate report
Detector Optimization Negative populations entirely on scale
Degeneration Increase SS Decrease FS 08-03307
Degeneration Decrease in intensity for many antigens 08-03307
Viability Gate 08-03307
Viability Gate All cells Viable cells 08-03307
Sample Exhaustion Air in system gives rise to many spurious signals Event gate to exclude non-real events
Laser Delay Fluidic instability - Monitor events over time to detect
Laser Delay Original Gated
Doublet Discrimination • Doublets = > one cell in laser simultaneously • High cell concentrations • Cell aggregates, sample preparation • High sample aspiration pressure • Doublets have composite properties • Can exclude using height, area, or width
Example Original 07-04513
Example Time 07-04513
Example Singlets 07-04513
Example Viable 07-04513
Determining Positivity Incorrect Correct 07-08661
Cell Type Identification Lymphocyte population identified by FS/SS gating
Cell Type Identification Borowitz et al (1993) AJCP 100:534-40. Steltzer et al (1993) Ann NY Acad Sci 667:265-280
Lineage Identification • CD19 for B cells and CD3 for T cells • Assumptions that may not always be correct • Always use at least two methods of identification
Normal B cell Maturation Wood and Borowitz (2006) Henry’s Laboratory Medicine
Follicle Center B cells Follicular Hyperplasia 08-03324 Follicular Lymphoma 08-01359
ALL MRD 0.1% abnormal immature B cells 06-01469
Data Analysis • Data displayed as dot plots or histograms • Restrict to subset having high informational content • Color discrete populations • Display information from other parameters • Allow rapid visual identification in multiple plots • Display data in consistent manner • Pattern recognition