Hi Jay, It is getting a bit too close to Christmas for me to think clearly but I'll do my best! If you are using the basic "two platform" ISHAGE i.e. using a hematology analyser to get your white blood count and the flow for the percent, you have a problem. Post thaw products are pretty much devoid of viable granulocytes and this increases the percent of CD34. If you are multiplying the post thaw CD34 percent by the pre (or even post processing WBC) you will obtain a falsely elivated absolute CD34 count. You have to use the single platform ISHAGE method which uses a fluorescent bead to allow direct calculation of absolute CD34 from the flow cytometer. In this way, it doesn't matter if you exclude 7-AAD+ dead cells from the assay, as while your percent CD34 will go up, the absolute number is a direct relationship to the number of CD34+ cells (on histogram 4) to the number of beads collected. You are in effect using the beads to determine the volume of sample collected and hence the number of CD34 cells per unit volume. Rob Sutherland and I have worked extensively on this methodolgy and will happily send you additional data should you require it. Rob uses BD equipment and I use Beckman Coulter (it keeps us honest!). In addition there are listmode files on the ISHAGE website at www.ishage.org - follow the committee links to graft evaluation. This site is still under construction but has the basic CD34 viabilty template with listmode files. Feel free to contact me directly if you need more information. I'm sure Rob is reading this also and he will be happy to respond too! Merry Christmas! Mike Keeney <<< <jglu@mail.mdanderson.org> 12/22 8:36p >>> In our BMT cell processing Lab, we routinely detect CD34 on PBPC after collection(fresh PBPC) and before infusion(thawed PBPC). We often find CD34 express in Thawed PBPC was higher than in fresh PBPC(same product). We use ISHAGE method to calculate the CD34. We also use 7AAD to exclude the dead cells. In the dot-plot(CD34PE vs SSC), we sometimes see denser or bigger population in thawed PBPC(compare to fresh same PBPC). Anyone has same experience? what are possible reasons to cause this? Is there any way to solve this problem and get more accurate % CD34 and recovery? Thank you for your precious opinion. Jay
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