Dean Lee, Kwon Ye Jin, Philpott M Katherine, Stanciu Cristina E, Seashols-Williams Sarah J, Dawson Cruz Tracey, Sturgill Jamie, Ehrhardt Christopher J
Department of Forensic Science, Virginia Commonwealth University, 1015 Floyd Ave, Richmond, VA 23284, USA.
School of Nursing, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA 23284, USA.
Forensic Sci Int Genet. 2015 Jul;17:8-16. doi: 10.1016/j.fsigen.2015.03.003. Epub 2015 Mar 12.
Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A02 allele. Minor peaks from the A02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.
对生物混合物进行分析是法医实验室面临的一个重大问题，尤其是当混合物中仅包含一种细胞类型时。多个个体对生物证据的贡献会使DNA图谱解释变得复杂，并且常常导致DNA证据的证明价值降低，甚至更糟的是，导致其完全丧失证明价值。为了解决这一问题，我们采用了一种分析技术，该技术利用个体之间固有的免疫差异，在进行DNA分析之前，从混合物中物理分离出来自不同来源的细胞。具体而言，我们应用了一种荧光标记的抗体探针，通过与大多数有核细胞表面表达的人类白细胞抗原（HLA）蛋白进行等位基因特异性相互作用，选择性地结合混合物中的一个贡献者的细胞。一旦贡献者的细胞与探针结合，就使用荧光激活细胞分选技术（FACS）将其从混合物中分离出来——这是一种基于细胞光学特性分离细胞群体的高通量技术——然后进行STR分析。我们在两人和四人全血混合物上测试了这种方法，其中一个贡献者拥有混合物中其他贡献者所没有的HLA等位基因（A02）。结果表明，混合物与与A02等位基因的蛋白质产物互补的荧光标记抗体探针杂交，产生了具有独特光学特征的细胞群体，该群体可以很容易地与混合物中的其他细胞区分开来。在用FACS对细胞进行分选后，基因分析表明，该细胞群体的STR图谱与拥有A02等位基因的贡献者的图谱一致。观察到来自A02阴性贡献者的微小峰，但可以很容易地与A*02阳性细胞产生的图谱区分开来。总体而言，这表明与FACS相结合的HLA抗体探针可能是从法医混合物中生成个体贡献者STR图谱的有效方法。
