Determination of platelet purity.

(A) Total cellular RNA (1.8 μg) from platelet-rich plasma (PRP) or purified platelets from a single apheresis donor were analyzed by RT-PCR (35 cycles) using oligonucleotide primers specific for glycoprotein IIb (GPIIb), T-cell receptor β-chain (TCRβ), or CD45; 10 μL of the 50 μL reactions were analyzed by ethidium-stained agarose gel electrophoresis. Minimal to no TCRβ gene product was visually evident only in PRP. Size markers corresponding toHaeIII-restricted φX174 DNA are shown. (B) Real-time RT-PCR was completed by using 1.8 μg total RNA and TCRβ-specific oligonucleotide primers optimized for quantitative analysis by real-time PCR.18 On the basis of parallel determinations using RNA isolated from known amounts of purified leukocyte standards, the leukocyte-depletion protocol represents an approximate 2.5-log purification from the starting PRP. Results are representative of one complete set of experiments repeated on 2 separate occasions, and data points represent the mean from triplicate wells, with standard errors of the mean (SEM) less than 1% (not shown).