Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group. The NIH/NIAID/DAIDS/ACTG Virology Laboratories

Abstract

An independent quality assurance program has been established by the Division of AIDS, National Institute of Allergy and Infectious Diseases, for monitoring virologic assays performed by nearly 40 laboratories participating in multicenter clinical trials in the United States. Since virologic endpoints are important in evaluating the timing and efficacy of therapeutic interventions, it is imperative that virologic measurements be accurate and uniform. When the quality assurance program was initially created, fewer than 40%o of the laboratories could consistently recover human immunodeficiency virus (HIV) from peripheral blood mononuclear cells (PBMCs) of HIVinfected patients. By comparing coculture procedures in the more competent laboratories with those in laboratories who were struggling to isolate virus, optimal conditions were established and nonessential reagents and practices were eliminated. Changes were rapidly introduced into a laboratory when experience dictated that * Corresponding author.

Infection by HIV‐1 blocked by binding of dextrin 2‐sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T‐cells

“…The ability of D2S to block infection of PBMN cells by primary viral isolates of HIV-1 was also determined. These isolates were grown from HIV-1 positive patients by culturing their PBMN cells (2 x 106 ml-') with an equal number of PHA activated PBMN cells from seronegative donors (Hollinger et al, 1992). The co-culture was fed every 4 days with fresh LGM containing IL-2 and fresh PHA activated PBMN cells (0.5 x 106 cells ml-').…”

Statistical design for a small serial dilution series

“…Similarly, many studies express N in terms of virions per unit volume, as opposed to our approach of treating N as an absolute number. The approximation p i ≈ 1−exp{−N i } is often used at (1) when N is known to be very large and i is small but we will not use this approximation.…”

“…This is approximately the number of aliquots that we will be concerned with but our results also hold more generally. Each stage of our prototype represents 1 3.84 concentration of the previous so d 1 =···=d 5 = 3.84. Laboratory pipettes are continuously adjustable so a fractional dilution rate does not pose a problem.…”

“…Conditional on N, the Y ={Y i } are jointly distributed as independent binomial random variables with respective parameters n ={n i } and p i (N) given at (1). A gamma prior distribution with density function (5) is assumed for the number of viral particles N>0 in the original solution for parameters >0 and >0.…”

“…The replication of HIV is monitored by determining the amount of viral products generated in culture, usually the quantification of the amount of the viral protein produced during viral replication [1] using an enzyme immunoassay. This technique employs enzyme-linked antibodies that bind specific viral proteins and produce, enzymatically, a colored product that is quantified spectroscopically.…”