Here is a synopsis of the responses to the CAP survey question: Question: We have difficulties setting the gates appropriately due to the quite altered scatter pattern of the preserved survey specimens. We also have issues with low white cell counts. Does anyone else have these or other problems? Do you have any suggestions for processing or analyzing that we could try? 1: The surveys have had very low white counts--I manufacture them. You might want to wash the cells in RPMI and concentrate the cells. This will work if you are not doing absolute counts. 2. We routinely use three color analysis, however when we have a sample that has red cells resistant to lysing, or an old shipped sample, we add CD45 APC. 3. I have been evaluating CAP Lymphocyte panel for several years and have not experienced any light scatter problems nor any gating problems. The only difference is that I report out %Total and have to calculate differently to report out %gated, however use the same analysis templates that we use for our patient sample (PB, BM, PBPC, UCB). I run CD Chex simultaneously as a control. We use a stain-lyse-wash technique and usually perform a cell count on the samples, adjusting to 1e7 per mL using 100uL per test. If counts are low, I use 100uL of sample and resuspend in 200-250uL for acquisition; acquiring 20,000 total events. 4. We live on the West coast of Canada and received our samples 7 days after the date that most of the participants received and ran their samples. The viability of our samples was not great and the scatter plots weren't either. We normally use FS vs. logSS and found the lymph population to have higher SS than the grans but with the usual FS. We did our best but had it been a clinical sample we would have been interpreting with much caution. We used isotypic controls whenever possible on both cases and CD45 vs. SS gating on FL3-04 (where scatterplot was difficult to interpret, viability was poor and we thought it might be a high grade lymphoma/leukemia). 5. We failed once many many years ago-someone did not transcribe the appropriate data from the graphics to the cap form correctly. I would check this first before pulling out the facscomp report from the day you ran them or looking at the raw data again... 6. The immunophenotyping cells are cell lines and are generally large. To date in all the proficiencies I see, there is only one cell line present so it is not too hard to adjust scatter settings to find them. It seems we have to accept that. I also feel that the survey for T4 etc are problematic with low counts. The low counts can be frustrating. 7. You're right, it shouldn't matter about instrument or panel and instrument and fluorochromes are taken into account when the samples are evaluated by CAP. If %positives are outside range, you might try looking at a dot-plot of CD3 vs. SSC (routine samples and CAP) to determine if you have any 'escapees'. This can happen, depending upon anticoagulant, fluorochrome combination and/or centrifugation speed. I minimized this problem by using CD45 FITC and CD3 PerCP and washing at 150g for 5min. Thank you all for your responses! We are doing much of what was suggested, but will try a few new things with the next survey, such as concentrating the specimen and viewing CD3 vs. SSC. I will also fax the graphs after the results come back for the next survey to anyone who would like to discuss it further. Thanks for your valuable time and input, Melanie O'Donahue, MT, ASCP Mayo Clinic Scottsdale Laboratory Medicine Flow Cytometry Lead Technologist Odonahue.Melanie@mayo.edu 480-301-8028
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