1 ITN clinical trial analysis design – Transplantation example Cross-sectional and longitudinal clinical trials to assess tolerance Disease areas: Transplantation Allergy & Asthma Auto-immune diseases Large studies: 120 per-protocol participants 10 time-points 9 panels = 10,800 FCS files Hypothesis (Allergy): Alterations in Cell Surface Chemokine Receptors (TH2  TH1 Shift) TH1: CD4+, CCR5+, CXCR3+ TH2: CD4+, CCR4+, CCR3+

2 Based on output from subjective gating  Assay Quality Assurance Assessing inter- and intra- assay variability within and across trial visits  Analysis Populations of interest: Subset data on domain expert’s interpretation Use standard parametric and non- parametric approaches Pattern visualization Quality AssuranceStatistical Analysis & Pattern Visualization

3 Concerns –Subjective gating Poor reproducibility –Variability among experts in defining populations of interest –Temporal effects may be confounded by laboratory processing –Limited software support for high-throughput flow cytometry statistical analyses Future directions –Pattern recognition approaches to un-gated or algorithmically gated data may define novel patterns –Large clinical trial datasets may be used for testing and validation of patterns