流式细胞(FACS)多次试验数据比较.pptVIP

Standardization Comparing Data Across Cytometers Scenarios include: Core facility: the same cytometer is not always available. Clinical trials done at multiple sites. Research collaboration between different laboratories and institutions. Sample Run on Different Cytometers Standardization Across Cytometers GOAL: To be able to compare application results across sites and platforms. NEED: A method to establish consistent and reproducible MFI targets across time and cytometers. Standardizing Setup with CST One Cytometer—Day to Day Use Performance Check to track instrument performance. Use Application Settings to position cells of interest at optimal target MFIs. Multiple Cytometers Use Performance Check to track instrument performance. Use Application Settings to position cells of interest at optimal target MFIs. Application settings standardize the target MFIs across multiple cytometers by using a reference particle for each parameter. Standardization Reference Particles When all cytometer optical configurations are the same: MFI Target Values for Standardization From the cytometer baseline reports, determine: the highest rSD of electronic noise for each parameter the lowest Linearity Max Channel for each parameter Select one cytometer and adjust the fluorescence parameter PMT voltages as needed to optimize for the cells of interest. On the selected cytometer, run the reference particle for each parameter. The MFI target values for standardization are the MFIs of the reference particles. Optimizing for Cells of Interest On the selected cytometer: Run unstained cells and for each fluorescence parameter, verify that the rSD of the negative population is about 2.5 times the highest rSD of electronic noise. Adjust PMT voltages as needed. For each fluorescence parameter, run cells stained with a relatively bright reagent and verify that the MFI of the positive population is less than 0.5 times the lowest linearity max channel. Lower the PMT voltage as n