Stain Method Applicable To Micromanipulation-LVPCR Technique And Its Forensic Application

Objective:The coordination of micromanipulation and LVPCR technique is an effective way to isolate and test mixed microscale forensic biological specimens. Due to the variation of cell quality and impurity contamination in case samples, whether or not the cell can be clearly identified under microscope become a key factor affecting the result of test. Based on a micromanipulation flat and the principles of histological dyeing, the author aims to establish a stain method applicable to micromanipulation-LVPCR technique and appraise its apply effect in forensic practices.Methods:Prepare cell suspension using swabs with oral epithelium cells from 10 volunteers and 2 real case samples; 0.05g/ml gentian violet solution and 2.5×10-4 g/ml methylene blue solution. Establish and optimize cell suspension stain method by comparing staining effects of gentian violet, hematoxylin and methylene blue solution under different concentration and time gradient; Micromanipulation carried out after the same dose of oral epithelium cells have been dyed respectively with three solutions to compare staining effect of each solution and define the best staining time, check their influence on final test result of LVPCR to establish a best stain method; Apply the optimized method to oral epithelium cell suspension drawn from volunteers and then capture and test the same number of stained and unstained cells from different specimens. After 20 duplications, success rates of the STR genotyping in cell micromanipulation were compared under the staining and non-staining condition to verify the influence of stain method on the result of LVPCR. The stain method had then been applied in oral epithelium cell micromanipulation in a criminal case to further validate its effect.Results:0.5μl 0.05g/ml gentian violet solution added into 100μl cell suspension for 5mins could distinguish the nuclei from cytoplasm by staining them into violet; no negative influence detected upon the extension of stain time (≤60min). All the three solutions can clearly stain the nuclei of oral epithelium cells under optimized condition——gentian violet solution being the favorable choice with markedly higher STR genotyping success rate, the extension of stain time (≤60min) having no detectable influence on DNA test result. STR genotyping success rate virtually not affected compared with micromanipulation result of the same dose of non-stained cells after duplicated tests to identical and different specimens (p>0.05). Nuclei could be clearly marked and easily spotted in case sample using this stain method and more cells can be captured to meet the standard of identical recognition after duplicated test.Conclusion:Gentian violet stain method is applicable in micromanipulation-LVPCR technique, which has a fundamental significance for the efficiency improvement of this technique and contributes to its further promotion in forensic practices.