Cytometry (Clinical Cytometry) 50:127–128 (2002)
Invited Commentary
Beckman Coulter and CD4ⴙ T Cells Enrique M. Rabellino, Meryl A. Forman, Grant C.J. Howes, Rhonda A. Mills, Jorge A. Quintana, Patricia A. Roth, and Julie G. Wilkinson Cytomics Center, Beckman Coulter, Inc., Miami, Florida
The significance of CD4⫹ T cells in patients with human immunodeficiency virus (HIV) infection/AIDS was first recognized in the mid 1980s when studies demonstrated a close correlation between CD4⫹ T-cell counts and HIV infection. Enumeration of blood CD4⫹ T cells soon became established as an undisputed clinical and laboratory surrogate marker for disease staging, prognosis, and subsequently, for the therapeutic management of HIVinfected patients. The drive to understand the pathogenesis of HIV infection has led to breakthroughs in our knowledge of the cellular mechanisms of the immune response including T-cell participation in antigen recognition and presentation, and cell signaling processes. In collaboration with basic and clinical researchers, clinicians, and engineers, the diagnostic industry has focused research and development efforts on serving the clinical needs of a growing AIDS patient population. Beckman Coulter has participated in the development of this field from the onset of the AIDS crisis. Our mission has been to promote both basic and clinical research by providing accurate and reliable reagents, as well as systems to simplify and automate methods and applications for clinical flow cytometry. The migration of CD4⫹ T-cell enumeration into the clinical laboratory was marked by the introduction of stable reagents, method controls, system standardization, and hardware advances. Beginning in the late 1980s, Beckman Coulter released a series of in Vitro Diagnostic Use (IVD) reagents for CD4 and CD8 enumeration that increasingly improved accuracy and simplified analysis. Gating techniques combining light scatter and CD45 along with CD3/CD4 and CD3/CD8 (three and four-color analysis) represented major achievements in the field. Development of an internal calibrator bead (Flow-Count™ Fluorospheres) allowed the direct measurement of absolute cell count on the flow cytometer, rather than a calculated count derived from a separate hematology platform measurement. This single-platform method led to a significant improvement in test precision. Digital signal processing technology on the EPICS威 XL™ flow cytometer represented another milestone and further increased accuracy by improving the analytical test linearity. Clinical flow cytometry also required quality control processes. Instrument standardization became practical with the development of calibration reagents for alignment, fluorescence, and color compensation. For assay control, CYTO-TROL™ Control Cells was the first human lymphocyte cell-based reagent used to control antibody-
© 2002 Wiley-Liss, Inc.
staining performance. With the introduction of IMMUNOTROL™ Cells (a stabilized preparation of whole blood components), the level of control was expanded to include the entire assay process. The addition of IMMUNOTROL™ Low Cells for low CD4⫹ T-cell count samples now provides control at the clinical decision level. Correlating control results across clinical laboratories has monitored the quality of laboratory performance. The Coulter Interlaboratory Quality Assurance Program (IQAP) was the first corporate-sponsored proficiency program for flow cytometry performance. Automation of flow cytometry started in the late 1980s with the introduction of many user-friendly functions, easy maintenance programs, and effective data management capabilities. The Q-PREP™/IMMUNOPREP™ workstation combined with the CYTO-STAT威 reagent product line first provided a rapid, no-wash method for whole blood immunophenotyping. The tetraONE™ System combined these automation advances with four-color flow cytometry and software for automated analysis, resulting in a system for rapid, standardized, and accurate singletube measurements. Furthermore, the consolidation of the multitube carousel loader with the cytometer added walk-away testing capabilities to the system. The next enhancement combined the TQ-Prep™/PrepPlus™ 2 workstation, which simplified and automated multiple sample processing by providing closed tube handling, and delivery of all reagents including specimens, antibodies, lytics, and controls as well as programmable incubation timing. In contrast to full automation, laboratories without ready access to flow technology require manual methods to generate CD4 results. To address this need, in the early 1990s, Beckman Coulter released an IVD microscopebased manual method, which identified CD4⫹ T cells by rosetting with monoclonal antibody-coated beads. Although CD4⫹ enumeration has been instrumental for patient and therapy management, there are a number of virological parameters that play essential roles in the comprehensive approach to diagnosis, therapy, and drug discovery in HIV. Detection of HIV p24 antigen was critical *Correspondence to: Enrique M. Rabellino, Cytomics Center, Beckman Coulter, Inc., 11800 S.W. 147 Ave. M/S 21-A01, Miami, FL 33116. E-mail:
[email protected] Received 15 February 2002; Accepted 15 February 2002 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cyto.10096
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for the initial development of antiviral therapies and the prevention of neonatal HIV transmission. In addition, it has been instrumental in the early detection of HIV infection and protection of blood supply. Measurement of HIV RNA in blood now provides the direct indicator of viral replication activity. Analysis of DNA to determine the prevalence and genetic diversity of HIV subtypes is also used to assess therapeutic efficacy. Although the enumeration and quantitation of CD4⫹ T cells remains one of the definitive surrogate markers for HIV disease management, it is apparent that a single measurement is not enough. Functional subsetting has become a critical issue, as the search continues for suitable surrogate markers for clinical response and true protection. New markers are required for the evaluation of emerging therapies as well as for monitoring established modes of treatment. Antigen-specific T-cell analysis is being used to measure immune response as new immunotherapy and vaccine programs enter clinical trials. Beckman Coulter has intro-
duced MHC tetramer reagents (i●TAg™) in order to gauge the magnitude, duration, and specificity of the anti-HIV– directed immune response. Phenotyping reagents that elucidate the effector capacity of both CD4 and CD8 T cells are also being developed. New markers and combinations have emerged to define memory and naive subsets, repertoire diversity, activation status, and Cytotoxic T Lymphocyte (CTL) function. These are also relevant to assess the synergistic effect of the immune response with antiviral drug therapies. New challenges in the diagnostic field will emerge as the understanding of HIV pathogenesis evolves. Basic discoveries on the mechanisms of immune recognition and function will lead to new perspectives on patient management and therapeutic approaches. Thus, evaluations of antigen-specific responses, cell effector functions, and regulation of signaling pathways are likely to play major roles in the development of vaccines and new antiviral drugs and in the optimization of current therapeutic strategies.
