2.1 Preparation of Plasma，Platelets，and Peripheral...
Whole blood（10 ml）was collected in acid citrate dextrose tubes.A platelet-rich plasma preparation（PRP）was prepared by centrifugation of the whole blood at 500 g for 15 min.The PRP fraction was further centrifuged at 6，000 g for 30 min and separated into a supernatant fraction（PFP or platelet-free portion）and a platelet pellet（PP）.The PP was resuspended to 1/10 of the original plasma volume with TE buffer.HIV-1 RNAs from all three fractions（PRP，PFP and PP）were extracted with RNAzol（9∶1，v/v）.
PBLs from seropositive patients were prepared from whole blood by standard Ficoll-Hypaque separation.The PBLs were washed in PBS twice，and total RNA was extracted with RNAzol.Normal PBLs were prepared by an alternate method to evaluate the potential of HIV DNA in plasma to contribute to the RT-PCR signal of the guanidinium extracted nucleic acids.One milliliter of fresh blood from a seronegative individual was mixed with 2 ml of Isoton Ⅱ and 20 μl of Zap-Oglobin Ⅱ lysing agent（Coulter Diagnostics，Hialeah，FL）at room temperature for 2 min，and centrifuged in a microfuge for 2 min.The white cell pellet was washed with Isoton Ⅱ and mixed with 800 μl of RNAzol that was adjusted either to pH 4.0 or 8.5 as described below.Then a standard，synthetic HIV-1 cRNA（105 copies）and a standard HIV-1 DNA（105 copies；linearized pBKBH10S）were spiked into this mixture.(https://www.daowen.com)
