Biosafety considerations for flow cytometric analysis of human immunodeficiency virus-infected samples.

Abstract

Since the recognition of human-immunodefiency virus (HIV-1, -2) infection as a separate disease entity, flow cytometry has been an invaluable tool for the study of the effects of these viruses on cellular parameters. As early as 1983, before the identification of the causative agents of the disease, immune abnormalities in circulating T lymphocyte subpopulations in patients affected with the Acquired Immunodeficiency Syndrome (AIDS) were noted (2,31). Most importantly, these patients presented with decreased numbers of CD4-bearing T lymphocytes; subsequently, CD4 cell frequencies as measured by flow cytometry were found to be highly prognostic for prediction of disease survival (32). Commercial availability of monoclonal antibodies to various cell surface antigens and the generation of different fluorochromes for multi-color flow cytometry provided the possibility of in-depth analysis of the alteration of immune cell subsets observed during HIVinfection (reviewed in 12,15) and has also led to the identification of alternate markers for disease progression (13,18). Thus, flow cytometers are utilized frequently in research and clinical laboratories to analyze HIV-infected samples. However, previous reports about the hazards associated with the use of flow cytometers have focused on cell sorting (10,11,21), because flow sorters generate droplets and aerosols during their normal operation and thus may expose operators to toxins or to pathogens contained in the samples to be sorted. Recently, the Biohazards Working Group of the International Society for Analytical Cytology (ISAC) has generated biosafety guidelines for cell sorting of unfixed, viable cells (30). The document contains recommendations for sample handling, operator training and protection, laboratory design, instrument setup and maintenance, and testing for instrument aerosol containment and is available in the full-text version on the internet from the webpage of the ISAC Biosafety Committee at http://www.isac-net.org/committees/biosafety/biosafety.html. The general recommendations as set forth in the ISAC biosafety guidelines are also applicable to cell sorting of samples known to contain HIV. In contrast, the current document focuses specifically on the practical safety aspects of flow cytometric analysis of HIV-infected samples and addresses areas that require special attention in laboratories which process known biohazardous samples and analyze them on flow cytometers.