acquired on the flow cytometer. Because the sample was never washed, there is no opportunity for cell loss. It is of little consequence that the unbound antibody is still present in the sample; pulses caused when labeled cells are interrogated by the laser are measured relative to the background fluorescence of the sample stream (baseline) and thus are seen above the noise caused by unbound fluorochrome. During analysis, the internal standard beads are identified by their unique scatter and fluorescence profile. The number of bead events is counted and compared to the number of events of interest (e.g., CD45+ CD34+ cells). The absolute count is determined as the number of events of interest divided by the number of beads and multiplied by the known bead concentration. Given proper instrument performance and accurate gating of the beads and the events of interest, there are only two key steps in the process: The beads must be handled properly (brought to temperature and gently mixed) to assure that the bead concentration added is the same as that stated by the manufacturer (usually about I X 106/mL). The same pipetting device should be used for beads and sample, because the precision of volume measurement is critical (see Chapter 2, Statistics, for a discussion of precision versus accuracy). A positive displacement pipette is best for this purpose. Note that the volumes of antibody and lysing solution do not enter into the absolute count equation and are therefore not critical. Beyond relying on the bead concentration stated by the manufacturer, bead concentration can be independently confirmed on a hematology counter. An example of the single-platform method is given in Section 6.16. 6.13 QUALITY CONTROL FOR QUANTITATIVE FLUORESCENCE Flow cytometers are so linear and perform well over such a large dynamic range that it is natural that they should be used for making quantitative measurements of proteins and other molecules detected on single cells. The problem has always been one Und<'rslanding Clinical flow Cylornetry 207 of calibration. How many molecules do you have on a cell when they have a particular MFI'? The fluorescence ofC04+ T-cells can be used as a poor Jl/Oll 's quantitative standard. providing that (I) normal peripheral blood mononuclear cclls, which are known to express on average 50.000 C04 molecules per T-celll60J, are used as the standard: (2) antibody conjugation is well controlled, sueh that antibodies being compared have identical fluorescence to protein ratios (PE is easiest, since a well-conjugated antibody has one PE molecule per antibody molecule); and (3) the antibodies are used at epitope saturating concentrations. The number of molecules of interest can then be calculated by taking the ratio of the geometric MFl of the molecule of interest divided by that of the C04+ population and multiplying by 50,000 [6l]. A more elegant and better-accepted method involves the use of a series of multipeak beads calibrated in terms of molecules of equivalent soluble fluorescence (MESF) units (Quantum Beads®, Bangs Laboratories). The beads are run daily and a linear regression is performed on geometric MFI of the beads versus known MESF. The MESF of the experimental sample is then determined by plugging the MFI of the unknown into the regression equation. The measurement of ZAP 70 expression in chronic lymphocytic leukemia provides a clinical example of the utility of quantitative fluorescence as a prognostic indicator. An excellent paper by Chen et al. [62] compares several methodologies, including quantitative fluorescence determined as molecule Clinical Applications of Flow Cytometry Section Scientific Communications 4 Downloaded from https://academic.oup.com/labmed/article/31/2/97/2657057 by guest on 04 December 2021 98 LABORATORY MEDICINE VOLUME 31, NUMBER 2 FEBRUARY 20 0 0 number can refer to both the antibody and the antigen. Therefore, FITC-CD3 indicates a monoclonal antibody against the CD3 antigen, which is conjugated to fluorescein isothiocyanate (FITC) to make it fluoresce green when excited with blue light. On the other hand, CD3+ T lymphocytes are the subpopulation of lymphocytes bearing the