I felt some defense of using beads for calibration of quantitative antigen density measurement is in order. I agree with what was so succinctly written by Bob Ashcroft and Deb Bergland regarding the assumptions inhererent in deriving an accurate or true quantitation of molecules/cell using bead standards. Additional problematic issues exist for the cell surface vs. total cellular measurements of those molecules that are associated with cell activation related changes, such as CD11b in neutrophils or CD62P in platelets. However, the methodology in most situations does allow for relative simplicity and excellent precision in quantitative cytometric measurements, typically with CVs of <5% in replicate samples. Thus, although the the quantitative measurement may not be absolute truth, the results can be derived both practically and precise in a manner that is highly correlated with a cell-associated molecular density. This assay performance makes it highly useful for both research and clinical applications (laboratory medicine is full of examples of assays where the normal range evolve with technologic improvements) of quantitative cytometry (flow and image). So from my perspective, I see an increasing use of bead calibration for quantitative cytometry, particularly if both users and vendors of such products acknowledge their limitations and define conditions for optimized useage. I am optimistic that the current world-wide efforts towards concensus use of bead standards will provide potential users wth further improvement of this technique and better commercial products. Bruce H. Davis, M.D. Wm Beaumont Hospital Royal Oak, MI USA
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