評估流式細胞儀檢測混合精斑檢體之成效

在性侵害的刑事案件實務上，混合檢體條件均不甚理想，特別當少量男性精子細胞處於大量女性上皮細胞的背景下，更增加鑑驗的困難度。再者，為處理各種腐敗檢體，如何研擬準確性高及分離細胞效果佳的技術，均是鑑識人員所必須克服問題之一。而流式細胞儀具有分離細胞特性，且針對特異性高的細胞更能儘可能收集，以上特性正適合用來檢測混合精斑檢體。本研究擬以女性口腔上皮細胞混合男性精子細胞，模擬混合精斑檢體進行流式細胞儀分析，依流式細胞儀前散射（forward scatter, FSC）與旁散射（side scatter, SSC）參數，可發現混合檢體中有2群細胞（分別位於R1及R2區），顯示流式細胞儀確實可將混合檢體進行分群。此外，為使細胞分群效果顯著，本研究另外評估數種抗體，以提升細胞分群效果。本研究選用男性精子細胞CD52、ACRV1、MOSPD3、JLP、DEFB126、PH20與ADAM23等抗體進行標定，結果顯示CD52抗體標定精子細胞效果最佳（R2區陽性比例88.7%）。女性口腔上皮細胞選用CD324、CD326、CD227等抗體，結果顯示這3種抗體標定女性口腔上皮細胞的效果均一致（R1區陽性比例分別為92.6%、91.4%及92.2%）。另以不同濃度男性精子細胞進行流式細胞儀分析，發現精子細胞濃度在10^3以下，精子細胞陽性訊號過低，恐會影響後續DNA型別分析。因此，本研究除使用表面抗原之差異外，亦利用不同參數（如核酸套數差異及排除CD45的訊號等），以提高精子細胞分群效果，為提升檢測混合精斑檢體成效，奠定重要基礎。

關鍵字

流式細胞儀；性侵害；精斑鑑定；精子細胞；口腔上皮細胞

並列摘要

It is difficult to identify sperms in the mixed semen specimen in which few male sperm cells are generally mixed up with a large number of female epithelial cells in sexual assaults. In addition, it is one of the problems that the forensic officers must overcome about how to develop a technique to separate cells highly accurately and efficiently in deal with decomposed samples. The flow cytometry is a technique used to separate and collect single cells in high specificity, and is an ideal tool for detecting the mixed semen specimen. In this study, we showed that the mixtures of male sperms and female oral epithelial cells which were mimic the mixed semen specimen were separated to 2 groups of cells (located in the R1 and R2 regions, respectively) by the flow cytometry technique through the parameters of forward scatter (FSC) and side scatter (SSC), indicating that the flow cytometry technique can indeed group the cells from the mixtures. Moreover, antibodies, including anti-CD52, anti-ACRV1, anti-MOSPD3, anti-JLP, anti-DEFB126, anti-PH20, and anti-ADAM23 for male sperm cells, and anti-CD324, anti-CD326, and anti-CD227 for female oral epithelial cells, were further applied to flow cytometry to obtain groupings more significantly. The results showed that anti-CD52 antibody was the best for labelling of male sperm cells (88.7% positive rate in the R2 region), whereas all of anti-CD324, anti-CD326, and anti-CD227 antibodies were comparable for labelling of female oral epithelial cells (the positive ratios in R1 area were 92.6%, 91.4%, and 92.2%, respectively). Mixtures with different amounts of male sperm cells were used to exam the limitation of the flow cytometry technique. When the amounts of sperm cells were below 10^3, the positive signal of sperm cells was too low and may probably affect the subsequent DNA type analysis. This study also used different parameters (such as differences in the ploidy of DNA of cells, and elimination of the CD45 signals, etc.) besides antibodies to improve the sperm cell clustering effect. Thus, this study established an important foundation for improving the effectiveness of sperm identification in mixed semen specimen.

並列關鍵字

Flow Cytometry ； Sexual Assault ； Identification of Semen ； Sperm Cells ； Oral Epithelial Cells

參考文獻

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評估流式細胞儀檢測混合精斑檢體之成效

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