TABLE 5.

Recommendations for handling of blood samples to be assayed for HIV-1 RNA with U.S. and international research cohorts

Area receiving recommendation	Research studies with U.S. cohorts	Research studies with international cohorts
I. Research priorities for HIV RNA testing	1. Validate use as a predictor of disease progression in pediatric patients	1. Infant HIV diagnosis for assessment of early efficacy results from perinatal prevention trials
	2. Evaluate as a diagnostic assay for HIV infection in young infants	2. Measurement in plasma, cervical-vaginal secretions, and breast milk to evaluate mechanisms of perinatal HIV-1 transmission
		3. Study of breakthrough in vaccine recipients
		4. Pathogenesis studies of cofactors and clade differences related to disease progression
II.  Component of blood used, specimen collection tube	1. Plasma is preferable to serum, but consistency is most important	1. Both cells and plasma be collected and stored
	2. Tubes containing EDTA were preferable to those containing ACD, followed by those containing heparina	2. Ideally, at a minimum samples would be collected in two tubes: (i) a tube containing EDTA would be used for plasma RNA and flow studies; (ii) tubes containing CPT, heparin, or ACD would be used depending on the research study and on-site laboratory capabilities.
III. Timing of specimen processing	1. Process (i.e., spin and separate) whole blood between 3 and 6 h after collection, particularly if the specimen is collected in a tube containing ACD	1. Ideally, the separation of plasma and cells was recommended to be done between 4 and 6 h but up to 24 to 48 h after specimen collection
	2. For specimens in tubes containing EDTA or other anticoagulants, processing could occur up to 24 h after collection
IV. Stability and storage conditions	1. For whole blood, store the specimen at RT for up to 24 h if culture or lymphocyte function assays are not planned; otherwise, store at 4°C for up to 24 h until processing.	1. Both cells and plasma should be frozen and stored in liquid nitrogen, given problems with electricity outages and −70°C freezers
	2. For plasma specimens, store at 4°C for up to 5 days, although storage at −70°C indefinitely is ideal, followed by storage at −20°C for up to 20 wk
V. Shipping conditions	1. Ship whole blood or plasma specimens within 24 h of sample collection; temperature extremes should be avoided by using an insulated container to pack and ship the specimens; freezing of whole blood should be avoided to preserve CD4 cells	1. Shipments should be on dry ice
	2. Plasma specimens can be sent at RT or 4°C or frozen
VI. Future studiesb	1. Future technical studies should focus on use of dried plasma spots for quantitative RT-PCR in plasma	1. Separation techniques applicable to developing country settings need to be validated
	2. Field deployable rapid diagnostic tests	2. Future technical studies should focus on the use of dried plasma spots for quantitative RT-PCR in plasma
		3. Development of assays that will detect multiple HIV clades well and a standardized panel of different clades of viruses for testing the assays

^aIf a silica extraction method for RNA is used prior to testing by the Roche Amplicor Monitor assay, then there may be less concern regarding which anticoagulant is used. 

^bAlso see research priorities.