Diagnosing and monitoring of patients with HIV/AIDS and other cellular immunodeficiencies often depends on lymphocyte immunophenotyping. This procedure has benefited from a number of advances in the past decade, including the development of multicolor flow cytometers and associated software for absolute lymphocyte enumeration.In 2004, the FDA cleared the six-color FACSCanto flow cytometer manufactured by BD Biosciences of San Jose, Calif. With the associated FACSCanto and FACSDiva software packages, the flow cytometer can identify and enumerate leukocyte subsets for in vitro diagnosis and monitoring of HIV/AIDS.In the September issue of Cytometry Part B, researchers from the Miller School of Medicine at the University of Miami and from Borinquen Healthcare Center, both in Miami, and from the University of Florida in Jacksonville reported that they compared the performance of the FACSCanto and the FACSCalibur, a four-color flow cytometer from the same manufacturer, as well as their respective software packages at enumerating lymphocyte subsets in HIV-infected patients.Four colors or six?They conducted the study because they already had one of each instrument in the lab. “The idea was to see how comfortable we are in case one of them breaks down, to see whether the information we obtain from these instruments is [in] any way different,” said Deshratn Asthana, principal investigator of the study. The six-color flow cytometer provides signal retention on six fluorescent and two scatter parameters. It includes digital electronics for processing as many as 10,000 events per second and a novel sample injection tube that supports carryover of less than 0.1 percent. The instrument uses an air-cooled 20-mW 488-nm solid-state laser and a 17-mW 633-nm HeNe laser. The excitation beams are transmitted to separate spots in the flow cell and, when particles intersect with the beams, a gel-coupled lens collects the optical signals at the back side of the flow cell. The scattered light and fluorescence signals are then transmitted to the detector arrays, which are arranged in an octagon for the 488-nm signals and in a trigon for the 633-nm signals. A serial reflective design enables collection of the dimmest emission signals first, beginning with the longest wavelengths and moving on to the shortest, further enhancing the sensitivity of the system.The instrument’s two software packages include the FACSDiva, with acquisition and analysis tools for research applications, and the FACSCanto software, intended for clinical applications in the area of immunophenotyping.Importance of softwareThe researchers tested the flow cytometers’ performances by measuring the expression of several cell surface markers on lymphocytes in the whole-blood samples of 66 HIV-infected patients submitted for routine lymphocyte immunophenotyping. They acquired white blood cell counts and the percentage of lymphocyte in the samples using a five-part differential hematology analyzer. They analyzed the samples on the four-color system using FACSCalibur CellQuest Pro software (version 5.2), and on the six-color system using FACSCanto (version 1.0.3) and FACSDiva (version 4.1) software; all of the software packages are produced by BD Biosciences. To maintain consistency during the experiments, the investigators used a single operator and took advantage of automatic compensation settings during instrument setup.The measurements showed that the enumeration of lymphocyte subsets can vary with the instrument and the software used. Specifically, the researchers observed notable differences between data acquired with CellQuest Pro and FACSDiva software on the one hand and FACSCanto software on the other. This finding suggested that six-color lymphocyte immunophenotyping should be performed with FACSDiva rather than with FACSCanto, and that laboratories should provide information on both the instrument and the software used, even for routine flow cytometry result reporting.Contact: Deshratn Asthana, Miller School of Medicine at the University of Miami; e-mail: dasthan@med.miami.edu.