Department of Biomedical Laboratory Science, College of Health Sciences, Eulji University, Republic of Korea; Department of Senior Healthcare, BK21 Plus Program, Graduate School, Eulji University, Republic of Korea.
Department of Neural circuits Research group, Korea Brain Research Institute, Daegu, Republic of Korea.
Forensic Sci Int. 2022 Jul;336:111348. doi: 10.1016/j.forsciint.2022.111348. Epub 2022 May 21.
Dried bloodstains at crime scenes provide abundant information for analyzing criminal identity of victims or suspects, morphological characteristics, and biological and chemical compounds. Therefore, they are considered important evidence by investigators at crime scenes. Moreover, the age of bloodstains can be used to determine the timeline of incidents at crime scenes; Inappropriately handled bloodstains may cause degradation of blood components. In this study, we identified a novel marker, hemoglobin subunit beta protein, as an internal standard to determine the age of bloodstains at crime scenes. We found that the target spot between 20 and 30 kDa in two-dimensional electrophoresis gradually increased in size. The hemoglobin subunit beta protein was identified from this spot using liquid chromatography-tandem mass spectrometry and verified using western blotting. Sample bloodstains were exposed to various environmental conditions (humidity: 30%, 60%, 90% at room temperature [RT]). Furthermore, the hemoglobin subunit protein extracted from the sample bloodstains at various time points (0 h to 30 d) was dissolved in our newly developed buffer solution and in deionized or distilled water. We also analyzed the expression levels of the protein in the sample bloodstains, dried at RT and under various humidity over time, using western blotting. In addition, we evaluated the protein extraction capacity of deionized or distilled water and the newly developed buffer from the sample bloodstains over time. At RT and 60% humidity, using the newly developed buffer, the hemoglobin subunit beta protein levels showed a gradually increasing pattern. Finally, we quantitated human hemoglobin subunit beta protein using western blotting and enzyme-linked immunosorbent assay, which revealed significant differences among the samples. In particular, the time points from 36 h to 30 days were considered for analysis. Thus, the hemoglobin subunit beta protein dried at RT and 60% humidity and further dissolved in the newly developed buffer solution can be used to determine the age of bloodstains at crime scenes.
现场干燥血痕为分析受害者或嫌疑人的犯罪身份、形态特征以及生物和化学化合物提供了丰富的信息。因此，它们被现场调查人员视为重要证据。此外，血痕的年龄可用于确定犯罪现场事件的时间线；如果处理不当，血痕中的血液成分可能会降解。在这项研究中，我们确定了一种新型标记物，即血红蛋白亚基β蛋白，作为确定犯罪现场血痕年龄的内参。我们发现，二维电泳中 20-30 kDa 之间的靶点逐渐增大。使用液相色谱-串联质谱法从该点鉴定出血红蛋白亚基β蛋白，并使用 Western blot 进行验证。将样本血痕暴露于各种环境条件（湿度：室温下 30%、60%、90%）下。此外，从不同时间点（0 h 至 30 d）提取的样本血痕中的血红蛋白亚基蛋白溶解在我们新开发的缓冲液和去离子或蒸馏水中。我们还使用 Western blot 分析了随时间在 RT 下和不同湿度下干燥的样本血痕中蛋白质的表达水平。此外，我们还评估了去离子或蒸馏水中和随时间推移从样本血痕中提取的新开发缓冲液的蛋白质提取能力。在 RT 和 60%湿度下，使用新开发的缓冲液，血红蛋白亚基β蛋白水平呈逐渐增加的趋势。最后，我们使用 Western blot 和酶联免疫吸附测定法定量检测了人血红蛋白亚基β蛋白，结果表明样品之间存在显著差异。特别是，分析了 36 h 至 30 天的时间点。因此，在 RT 和 60%湿度下干燥并进一步溶解在新开发的缓冲溶液中的血红蛋白亚基β蛋白可用于确定犯罪现场血痕的年龄。
