1. Protective Immunity Project Chris Larsen, MD, DPhil, PI Christine Martens, Ph.D. Project Manager Vicki Hertzberg, Ph.D. Director of Data Management and Analysis Core

2. 3 studies 2 human –New renal transplants (n=60) and 20 controls, followed for 2 years –Existing renal transplants (n=60) and 30 controls, receiving flu vaccine, followed for short time 1 macaque study, 30 macaques randomized to 6 groups

3. Longitudinal studies IDTime 1Time 2…Time t 1 … n

4. Measurements At each time point for each subject obtain measurements from: –Flow cytometry –ELISPOT/ELISA –M-array

5. Flow cytometry For each subject at each time, take several blood samples. For each blood sample, label all of the PBMCs with 8-10 markers (a panel). Cells will fluoresce according to the amount of each marker present. Apply 6-8 panels for each subject at each time. Each panel usually has 2 markers that are common to all other panels

6. What to the data look like? X-Y graph for fluoresence intensity for marker X vs marker Y. Gates are drawn that –Separate present from absent –Isolate cells of different types

7. Issues? How to analyze? Can we deal with fluorescence intensity rather than presence or absence? How do we institute QC for the gating?