New trends in affordable CD4+ T-cell enumeration by flow cytometry in HIV/AIDS

Abstract

Inexpensive antiretroviral therapy (ART) might soon be available to treat human immunodeficiency virus (HIV) infections in resource-restricted areas of the globe. The number of CD4+ T-cells in the blood is the single most important laboratory parameter to select patients for therapy at the right time and to monitor the effect of ART. The question is asked whether flow cytometry is adaptable to change from an expensive and complicated scientific analytical instrumentation to become a practical diagnostic tool that can be widely operated. Recent studies indicate that a branch of clinical/practical flow cytometry is gaining a new identity by a confident simplification and improvement of clinical protocols. These new observations can be marshaled into three new areas of development. First, the changes in the choices of reagents and staining protocols have been initiated by the preferred use of primary immunological gating during the flow-cytometric analysis, exploiting the exquisite discriminating power of antibodies. The recent National Institutes of Health (NIH) and Centers for Disease Control and Prevention (CDC) guidelines introduced CD45-assisted protocols that are also at the heart of the PanLeucogating protocol that revitalized the testing of absolute CD4 counts on double-platform. With CD45 staining the leucocyte populations can be reliably studied in aging samples. It is now also documented that minimal technology, that is primary gating with CD4 (or CD8) antibodies, provides excellent CD4 (or CD8) T-cell counts. Consequently the optimal affordable protocol uses CD45 and CD4 antibodies in combination. The second area of development is the absolute counting facility on flow cytometers. Most frequently, microbeads are added to blood to define the sample volume in instruments that are not equipped with a volumetric microsyringe. The beads are, however, expensive, and the utilization of the stable flow rate of the excellent instruments might soon provide an answer to replace dear microbeads. With stable flow, the time span of analysis tells the sample volume and the absolute counts can be calculated. Finally, stabilized blood cell standards and blood stabilizing fluid, referred to as Transfix, have recently been introduced for quality assurance. These products are important to prove the precision and accuracy of the new affordable protocols and also to check, and guide if necessary, the performance of the laboratories even in remote areas. These recent developments provide evidence about the renaissance of affordable flow cytometry, a precise, costeffective and quantitative technology that is capable of providing CD4+ T-lymphocyte counts in as high volume as >400 patient assessment per day at the fraction of the cost of the current Western prices. These recent achievements need to be taken into consideration when alternative, nonflow CD4-counting methods are assessed.