Thorogate Richard, Moreira Joana C S, Jickells Sue, Miele Margherita M P, Daniel Barbara
King's College London, Department of Forensic Science and Drug Monitoring, London, UK.
Forensic Sci Int Genet. 2008 Sep;2(4):363-71. doi: 10.1016/j.fsigen.2008.06.003.
Full DNA profiles can be generated from just a few cells; however these profiles can be contaminated from other cell types present at the crime scene. We report here on the development of an immunofluorescent technique to spatially locate human-specific blood in situ and also on the ability of this technique to detect individual leukocytes and the DNA contained within them. Four monoclonal mouse anti-human antibodies were evaluated; anti-glycophorin A to detect erythrocytes and anti-CD45, anti-myeloperoxidase (MPO) and anti-histone H1 to detect the nucleated leukocytes. Each antibody was labeled with either Alexa Fluor 488 or 568 for direct application to blood smears which allowed the simultaneous detection of erythrocytes and leukocytes. Furthermore, because histones are DNA binding proteins, the application of anti-histone H1 allowed the detection of DNA within a blood smear. Importantly it was found that full DNA profiles could be achieved after using this method with similar peak area ratios compared to untreated cells. The fluorescent antibodies were found to be human-specific with the exception of anti-histone H1 due to its conserved sequence. However, used in combination with anti-CD45 or anti-MPO the location of DNA from human-specific leukocytes could be detected. The technique was also tested on older blood stains and was still found to be sensitive and cell-specific after 4 months. Following the optimization of the methodology, the fluorescent antibodies were applied to short lengths of black cotton fibres covered with human blood spots. Although the background fluorescence from the cotton was found to be high, erythrocytes and even individual leukocytes could easily be detected, indicating that this technique could be used to detect extremely minute amounts of blood. Used in combination with laser capture microdissection (LCM), this method could be used to pick off individual leukocytes for LCN DNA techniques.
仅从少量细胞就能生成完整的DNA图谱；然而，这些图谱可能会被犯罪现场存在的其他细胞类型污染。我们在此报告一种免疫荧光技术的开发情况，该技术可在原位对人源特异性血液进行空间定位，还报告了该技术检测单个白细胞及其所含DNA的能力。评估了四种小鼠抗人单克隆抗体；抗血型糖蛋白A用于检测红细胞，抗CD45、抗髓过氧化物酶（MPO）和抗组蛋白H1用于检测有核白细胞。每种抗体都用Alexa Fluor 488或568标记，可直接应用于血涂片，从而能够同时检测红细胞和白细胞。此外，由于组蛋白是DNA结合蛋白，抗组蛋白H1的应用使得能够检测血涂片中的DNA。重要的是，发现使用该方法后可获得完整的DNA图谱，其峰面积比与未处理细胞相比相似。除抗组蛋白H1因其序列保守外，发现荧光抗体具有人源特异性。然而，与抗CD45或抗MPO联合使用时，可检测到人源特异性白细胞中DNA的位置。该技术还在陈旧血迹上进行了测试，4个月后仍发现其具有敏感性和细胞特异性。在对方法进行优化后，将荧光抗体应用于覆盖有人血斑的黑色棉纤维短片段上。尽管发现棉纤维的背景荧光很高，但仍能轻松检测到红细胞甚至单个白细胞，这表明该技术可用于检测极微量的血液。与激光捕获显微切割（LCM）联合使用时，该方法可用于挑选单个白细胞用于低拷贝数DNA技术。
