Tau KR, Witty DS, Guinchard B, Michel-Treil V, Campbell WF; International Conference on AIDS.
Int Conf AIDS. 2000 Jul 9-14; 13: abstract no. MoPeA2133.
K. R. Tau, Covance CLS, 8211 SciCor Drive, Indianapolis, IN 46214-2985, United States, Tel.: +1 317 273 79 39, Fax: +1 317 273 79 90, E-mail: kim.tau@covance.com
As clinical trials continue to examine therapies which drive the plasma viral load to undetectable levels, other reservoirs of HIV-1 are of potential importance in determination of efficacy. Peripheral blood mononuclear cells (PBMCs) are an important reservoir of HIV-1 proviral DNA and viral RNA. Amplicor HIV-1 Monitor version 1.5 (Roche) primers can detect cell-associated HIV-1. However, the sample preparation available in the Amplicor kit has not been validated for use with PBMCs. The Boom technique (Organon-Teknika) has been validated for extraction of a broad spectrum of specimen types, including PBMCs. The performance of coupling the Boom technology for sample preparation of PBMCs to quantitation of cell-associated HIV-1 by Amplicor HIV-1 Monitor version 1.5 was examined. PBMCs were spiked with a known number of 8E5 cells (a lymphoblastic cell line which contains a single defective proviral copy of HIV-1) and frozen at -70C. A linearity analysis was performed on four separate runs. The expected range was log 2.08 copies/ million cells to log 6.29 copies/ million cells . The observed values were log 2.47 copies/ million cells to log 6.29 copies/ml (r = 0.9423, x/y = 0.957). Inter-assay precision was determined by performance of a 4x5 panel on five days with two levels of controls. For the low level, the mean was log 4.40 copies/ million cells (SD = 0.26, CV = 5.9%). For the high level, the mean was log 5.87 copies/ million cells (SD = 0.11, CV = 1.8%). Intra-assay precision was determined by assaying a mid-level control 8 times on a single run. The mean was log 5.06 copies/ million cells (SD = 0.14, CV = 2.7%). Preliminary data indicate that the sensitivity of this assay is log 2.50 copies/ million cells. A global correlation with Covance CLS in Geneva is currently underway. Use of the Boom technique for extraction of PBMCs, followed by detection and quantitation of HIV-1 by PCR is a viable method for detection of cell-associated HIV-1 in AIDS clinical trials.
Publication Types:
Keywords:
- Acquired Immunodeficiency Syndrome
- HIV-1
- Leukocytes
- Leukocytes, Mononuclear
- Polymerase Chain Reaction
- RNA, Viral
- Reagent Kits, Diagnostic
- Sensitivity and Specificity
- Viral Load
- instrumentation
Other ID:
UI: 102237680
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