Platelets, a typical source of error in real-time PCR quantification of mitochondrial DNA content in human peripheral blood cells.

摘要: Nucleoside analogues can induce toxic effects on mitochondria by inhibiting the human DNA polymerase-gamma. The clinically observed toxicities range from slightly increased serum lactate levels to potentially severe and fatal lactic acidosis. A growing interest exists for detection of changes in mitochondrial (mt) content patients receiving antiretroviral therapy (HAART). Most studies use peripheral blood mononuclear cell (PBMC) fractions investigate mt via Real-Time PCR patients, not accounting platelets falsifying (mt)DNA:nuclear (n)DNA-ratio. In this study we suggest a procedure eliminate disturbing totally. 8 healthy controls (G1), 6 therapy-naive HIV-infected (G2) 9 under HAART (G3) were examined mtDNA:nDNA-ratio using technology. Different collection and/or PBMC isolation strategies analysed variances outcome at examinations same donor. Using prepared whole specimens, mtDNA:nDNA-ratios showed no differences all investigated groups (G1, G2, G3). Comparing platelet-depleted G1 with G2 revealed reduction 22% (p = 0.128) steeper 40% 0.0036) comparing specimens G3. Scrutinising differently processed within themselves, versus platelet-containing difference 26% 0.0406), whereas comparison platelet-free led comparatively more distinct 35% 0.0089). effect was seen G3, where 32% 0.01) 42% 0.0011) decrease. Furthermore analysing each single patient relation different methods, minor fluctuation margin could be found PCR. PBMCs detection, correlation low clinical signs, like elevated or lipodystrophy, observed. Light-microscopic evaluation platelets, reinforced results. Our data demonstrate that first step sample collection/preparation is critical valid illustration ultra-sensitive tubes an easy low-cost alternative expensive sorting elimination platelets. measurement surrogate marker signs mediated HAART.