Isolation and Quantification of HIV From Lymph Nodes

This chapter describes a standardized microculture technique adapted from a peripheral blood mononuclear cells (PBMC) microculture assay to isolate and quantify HIV from lymph nodes. This quantitative lymph node microculture estimates the number of infectious cells per million lymph node mononuclear cells. The assay, as described below, is performed in two 24-well tissue culture plates using six fivefold dilutions. Each sample of patient cells is cocultivated with phytohemagglutinin-stimulated normal donor PBMC for 21 d. The supernatant from each well is assayed for HIV p24 antigen production by the standard HIV p24 enzyme-linked immunosorbent assay (ELISA). HIV-1 infectious titers are expressed as tissue culture infective dose (TCID₅₀ /10⁶ cells) according to Reed and Muench.