Optimising Leucocyte Recovery From Bloodstains For Fluorescent In-Situ Hybridisation and Laser Microdissection DNA Profiling

Abstract

Mixtures of blood can challenge DNA analysis as the profile of the minor contributor may be masked by the major contributor. Coupling the techniques, X/Y Fluorescent In-Situ Hybridisation (X/Y FISH) and Laser Microdissection (LMD), allows male and female leucocytes to be specifically identified and isolated from a blood mixture. DNA analysis of target cells can then produce single source DNA profiles from the male and female contributors. The effectiveness of these techniques is dependent on the recovery of intact leucocytes from bloodstains and the integrity of the DNA within the leucocytes. This study aimed to develop an optimised recovery technique for the maximum recovery of intact leucocytes from bloodstains for X/Y FISH labelling and LMD isolation. Addition of 0.02% Tween20 to Extraction buffer was found to assist in maintaining leucocyte integrity, and was also found to be a suitable moistening solution for the recovery of dried bloodstains and elution of leucocytes from swabs. Temperature had a great influence on leucocyte integrity. Drying of bloodstained swabs and recovery at 4°C significantly increased the recovery of intact leucocytes. Leucocyte integrity was also found to be influenced by environmental conditions. Exposure of bloodstains to humidity had the most detrimental impact on leucocyte integrity, and the least impact was observed for bloodstains kept at room temperature. Cotton swabs, were found to be two-fold more effective in uplifting intact leucocytes from a dried bloodstain and three-fold more effective at eluting intact leucocytes into buffer than rayon swabs. Lobind tubes were also found to further assist leucocyte recovery. Although an optimum recovery technique was determined, the compatibility of X/Y FISH and LMD DNA analysis of recovered leucocytes was unable to be demonstrated in this study. X/Y FISH labelling of intact leucocytes was unsuccessful with the current protocol, as only weak, non-specific signals were observed. Furthermore, no DNA profile was obtained from the 75 Haematoxylin and Eosin (H and E) stained leucocyte sample tested. This may have been due to inhibitory effects, of the H and E staining and any co-extracted heme, or insufficient viable DNA was present in the sample. Further optimisation of the current X/Y FISH and LMD DNA protocols is required before these methods can be applied to X/Y FISH labelling and LMD DNA analysis of mixed bloodstain case samples.