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

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