1. Department of Laboratory Medicine University of Washington
Data Analysis
Department of Laboratory Medicine
University of Washington

2. Data Analysis Assess data quality Identify populations
Remove artifacts
Identify populations
Compare with normal
Identify abnormal populations
Quantitate and evaluate immunophenotype
Generate report

3. Assess Data Quality

4. Detector Optimization
Negative populations entirely on scale

5. Degeneration
Increase SS
Decrease FS

6. Degeneration
Decrease in intensity for many antigens

7. Viability Gate

8. Viability Gate
All cells
Viable cells

9. Sample Exhaustion Air in system gives rise to many spurious signals
Event gate to exclude non-real events

10. Laser Delay
Fluidic instability - Monitor events over time to detect

11. Laser Delay
Original
Gated

12. Doublet Discrimination

13. 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

14. Example
Original

15. Example
Time

16. Example
Singlets

17. Example
Viable

18. Determining Positivity

19. Determining Positivity
Incorrect
Correct

20. Population Identification

21. Cell Type Identification
Lymphocyte population identified by FS/SS gating

22. Cell Type Identification
Borowitz et al (1993) AJCP 100:
Steltzer et al (1993) Ann NY Acad Sci 667:

23. 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

24. Compare with Normal

25. Normal B cell Maturation
Wood and Borowitz (2006) Henry’s Laboratory Medicine

26. Follicle Center B cells
Follicular Hyperplasia
Follicular Lymphoma

27. ALL MRD
0.1% abnormal immature B cells

28. 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