Hobbs Kallie J, Cooper Bethanie L, Dembek Katarzyna, Sheats M Katie
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.
Vet Sci. 2024 Aug 1;11(8):346. doi: 10.3390/vetsci11080346.
Cell-free DNA (cfDNA) is fragmented extracellular DNA that is released from cells into various body fluids. Previously published data from adult horses supports cfDNA as a potential disease biomarker, but also shows that direct measurement in plasma is inaccurate due to matrix effect. It is currently unknown whether a similar matrix effect exists in foal plasma. Given this, the objectives of the current study were to investigate foal plasma for potential matrix effect during fluorescence measurement of cfDNA using a Qubit fluorometer, and to determine whether neat and/or extracted plasma cfDNA concentrations are significantly different in healthy, sick non-septic (SNS) or septic foals. We hypothesized that matrix effect would interfere with accurate fluorescent measurement of cfDNA in foal plasma. Further, we hypothesized that mean extracted cfDNA concentrations, and/or extracted cfDNA:neutrophil ratio, would be elevated in plasma of septic foals compared to healthy or SNS foals. Cell-free DNA was measured in neat plasma, and following DNA extraction with a commercial kit, from 60 foals. Foal plasma exhibited both autofluorescence and non-specific dye binding, confirming matrix effect. However, even with extraction, no significant difference was found in cfDNA concentrations, or cfDNA:neutrophil ratios, between healthy (sepsis score ≤ 5), SNS (sepsis score 6-11 and negative blood culture), or septic (sepsis score ≥ 12 ± positive blood culture) foals. Our data show that matrix effect interferes with accurate Qubit measurement of cfDNA in foal plasma and supports previous findings that plasma cfDNA concentrations are not associated with sepsis diagnosis in foals. Further research is needed to better understand neutrophil function and dysfunction in foal sepsis.
游离DNA（cfDNA）是从细胞释放到各种体液中的细胞外片段化DNA。先前发表的来自成年马的数据支持cfDNA作为一种潜在的疾病生物标志物，但也表明由于基质效应，血浆中的直接测量不准确。目前尚不清楚马驹血浆中是否存在类似的基质效应。鉴于此，本研究的目的是使用Qubit荧光计在cfDNA荧光测量期间研究马驹血浆中潜在的基质效应，并确定健康、非感染性疾病（SNS）或感染性马驹的纯血浆和/或提取血浆中的cfDNA浓度是否存在显著差异。我们假设基质效应会干扰马驹血浆中cfDNA的准确荧光测量。此外，我们假设与健康或SNS马驹相比，感染性马驹血浆中提取的cfDNA平均浓度和/或提取的cfDNA：中性粒细胞比率会升高。对60匹小马驹的纯血浆以及使用商业试剂盒提取DNA后进行了cfDNA测量。马驹血浆表现出自发荧光和非特异性染料结合，证实了基质效应。然而，即使经过提取，健康（脓毒症评分≤5）、SNS（脓毒症评分6 - 11且血培养阴性）或感染性（脓毒症评分≥12±血培养阳性）马驹之间的cfDNA浓度或cfDNA：中性粒细胞比率也没有发现显著差异。我们的数据表明，基质效应会干扰马驹血浆中cfDNA的Qubit准确测量，并支持先前的研究结果，即血浆cfDNA浓度与马驹脓毒症诊断无关。需要进一步研究以更好地了解马驹脓毒症中中性粒细胞的功能和功能障碍。
