Platelets, a typical source of error in real-time PCR quantification of mitochondrial DNA content in human peripheral blood cells.
Platelets, a typical source of error in real-time PCR quantification of mitochondrial DNA content in human peripheral blood cells.
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血小板是人外周血细胞线粒体 DNA 含量实时 PCR 定量的典型误差源。
DOI：
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发表时间：
2004
影响因子：
4.2
通讯作者：
F. Goebel
中科院分区：
文献类型：
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作者：
B. Banas;B. Kost;F. Goebel
核苷类似物可以通过抑制人类DNA聚合酶γ诱导线粒体毒性作用。临床观察到的毒性可以从轻微升高的血清乳酸水平到潜在的严重和致命的乳酸酸中毒。人们对检测接受抗逆转录病毒治疗（HAART）患者线粒体（mt） DNA含量的变化越来越感兴趣。大多数研究使用外周血单核细胞（PBMC）分数通过实时荧光定量PCR检测患者的mt DNA含量，而不是计算血小板伪造线粒体（mt）DNA：核(n)DNA比率。在这项研究中，我们建议一种完全消除干扰血小板的方法。采用Real-Time PCR技术检测8例健康对照（G1）、6例初治期hiv感染者（G2）和9例HAART治疗期hiv感染者（G3）的mtDNA: ndna比值。对同一献血者进行不同的采血和/或PBMC分离策略，分析其结果的差异。采用全血标本制备的DNA，各组（G1、G2、G3） mtDNA: ndna比值均无差异。比较G1与G2的血小板耗尽PBMC部分的mtDNA: ndna比率显示，G1与G3的标本相比，减少了22% (p = 0.128)，更大幅度的减少了40% （p = 0.0036）。仔细检查组内不同处理的标本，在G2全血与含血小板的PBMC标本中，mtDNA: ndna比率的差异为26% (p = 0.0406)，而全血与无血小板的PBMC标本的比较，mtDNA: ndna比率的差异相对更明显，为35% （p = 0.0089）。在G3中也发现了同样的效果，与含血小板的PBMC相比，全血的PBMC降低了32% (p = 0.01)，与无血小板的PBMC相比，全血的PBMC降低了42% （p = 0.0011）。进一步分析每个患者与不同方法的关系，可以发现使用无血小板PBMC标本进行Real-Time PCR的微小波动幅度。使用无血小板PBMCs检测mtDNA含量，可以观察到低mtDNA: ndna比率与临床症状（如乳酸水平升高或脂肪营养不良）的相关性。光镜下对血小板进行评估，比较含血小板PBMC组分和血小板耗尽PBMC组分，增强了Real-Time PCR结果。我们的数据表明，血液样本采集/制备的第一步对于使用超灵敏实时荧光定量PCR技术有效说明hiv感染患者的mt DNA含量至关重要。使用血清管采集血液是一种简单和低成本的替代昂贵的细胞分选，以消除令人不安的血小板。使用无血小板PBMC组分测量mt DNA含量可作为HAART介导的临床体征的替代标记物。
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.