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On November 15, 2001, during the Centers for Disease Control's 3rd National Conference on CD4 ϩ T-Cell Immunophenotyping, in Orlando, Florida, a discussion focused on the consistency of sample delivery and the stability of clinical flow cytometers. These issues were raised in the context of an enquiry about the effectiveness of placing calibrator microfluorospheres (referred to as beads) into all samples during routine, absolute leukocyte enumeration using bead-based single platform technology (SPT). With SPT it is possible to generate absolute and percentage T-cell subsets from one specimen tube with the use of a non-volumetric instrument. A related concern was the reliability of results obtained with SPT, because the parameters generated with SPT rely exclusively on information generated from one tube, and this single measurement might be prone to error due to flow rate (FR) variation and might compare unfavorably with the precision of absolute counts derived from hematologic instruments, e.g., during the assays performed on double platforms. Interestingly, the answers to both dilemmas depend on the stability of the currently used flow cytometers. In particular, the participants at the Orlando meeting were asked to report any aberrant data pertaining to FR measurements during sample acquisition time (AT). THE HYPOTHESISThe FR during sample AT can be readily determined by analyzing the number of beads detected each second. If the delivery system of the flow cytometer is unreliable, then there might be a concern about the overall usefulness of the SPT protocol. Conversely, if the FR is constant, then the following assumptions should follow:1. Irregularities of FR in tubes will detect pipetting errors that may occur when beads are added to the specimens.2. The magnitude of pipetting errors will be directly proportional to the volume error. Thus the relation between FR and absolute cell count can be established.Based on this simple relation, an internal quality control (IQC) protocol can be incorporated (1). Such built-in procedures will monitor the quality of pipetting during immunophenotyping.3. With an empirically established FR value range, an internal error checking routine can be established.4. If the FR value is stable, the volume of the sample processed during an acceptable AT must be consistent. Consequently, although the role of beads remains important to verify the stability of FR, there is no need to add beads to each specimen. With the newly proposed SPT protocol, the use of beads is limited to calibration verifiers in a daily IQC procedure. This approach was established for clinical volumetric flow cytometers in 1995 (2). OBSERVATIONSDuring the conference, participants agreed that a study of SPT list mode files was a first step in establishing whether the FR with TruCount beads on Becton Dickinson Biosciences (BDB) flow cytometers and with FlowCount beads on Beckman Coulter (BC) instruments are consistent. We found that AT is not an automatically acquired parameter on BDB instruments.

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The single-platform absolute T-lymphocyte subset analysis was evaluated utilizing a universal protocol in a Canadian multicenter study with the collaboration of the members of the Canadian HIV Trials Network (CTN). Participants used flow cytometers and reagents of their choice for labeling and lysing whole blood. Over a 2-year period, CTN laboratories performed single-platform absolute T-lymphocyte subset enumerations on fresh and commercial stabilized blood products using commercially available microfluorospheres TruCount and Flow-Count. This multicenter evaluation demonstrated that the application of a universal template for single-platform analysis provides a generic approach that embraces a wide array of immunophenotyping settings available in clinical laboratory. Cytometry (Clin. Cytometry) 50:62-68, 2002.