I think we can agree that if 4/8 double positives exist in the periphery they are there in quite small numbers. And what their biological relevance is ( if any) I'll leave for the experts to sort out. But while we do not report the percentage or absolute number of these double positives into our data base, we do measure them as part of an internal QC check when running our many protocols. Frequently, we stain lymphocytes in whole blood with several antibody cocktails, each of which contains CD3 in combination with other markers of interest, including CD4 and CD8. We like to see the %CD3 value in each of several tubes to be within 3% of each other. We report the average of the %CD3 when applicable. We also sum the CD4 and CD8 values. If that sum is greater than 10% of the average CD3 value, we stain for Gamma/Delta T cells. The % gamma/ delta value is generally very close to the delta of the average CD3 minus the sum of the CD4+CD8. Conversely, if the sum of the CD4+CD8 gives a delta of -5% or greater, we stain for double positives. Again, generally speaking the % double positives will approximate (if not equal) the delta of the average CD3 minus the sum of the CD4+CD8. If these numbers do not "add up" as we expect we look to our process (staining technique, instrument problems, etc.) for answers before making any assumptions about the biological relevance of these cells. The fact is, when we have established that these cells are "truly" gamma/deltas or double positives they do not seem to cause a great stir in our laboratory. What do others of you think of our protocol and thinking in this regard? Fred Menendez
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