Single sperm cell isolation by laser microdissection.

In 1996, Van Oorschot and Jones firstly reported in scientific correspondence that short tandem repeat (STR) profiles could be obtained from cells left on different objects. Since then, forensic scientists have focused their efforts in isolating single cells as it can be extremely helpful in solving case works where sexual violence was concerned. Laser microdissection is a micromanipulation procedure allowing to cut off precisely the cells of interest from tissue samples or smears by a laser beam fitted with an optical microscope. We have harvested single sperm cells by laser microdissection using a Leica AS LMD (Leica Microsystems, Germany); laser setting, pulse laser intensity and laser alignment as well as recovery of the specimen have been properly fitted to the samples we were dealing with. Different tissue preservation, fixation, histological staining (Papanicolau, Nuclear Fast Red-Picroindigocarmine) methods and number of harvested cells for each sample have been evaluated as well. Finally, the genotype of sperm cells has been determined by STR typing, evaluating the sensibility of this forensic technique according to instrumental and biological above-mentioned variables.

Laser microdissection and DNA typing of cells from single hair follicles.

Isolation and identification of single cells from tissue samples or smears assume a great relevance in pathological and forensic applications; in this latter field, the possibility to identify a specific genetic profile can be obtained by short tandem repeat (STR) typing, allowing to achieve a scientific proof important in law courts. It is well known that DNA extraction may be performed from several tissue fragments, blood traces, spermatozoa as well as telogen hair. However, in the last case, few follicle cells are coupled to a great amount of keratin reducing the efficiency of DNA amplification. Recently, the introduction of laser microdissection technique has greatly improved the capability to select single cells without any cross-contamination. In the present report, we have performed a laser microdissection using a Leica AS LMD (Leica Microsystems, Germany), utilized on cutting the telogen hair in order to exclusively collect the lower part of the follicle and reduce keratin contamination. In this way we can accurately extract an adequate amount of DNA, successfully typed by STR profile.

A novel approach in personal identification from tissue samples undergone different processes through STR typing.

Short tandem repeats (STRs) or microsatellites have been recognized worldwide as a powerful tool for human identification. They have become widely used in human identification especially in criminal cases and mass disasters. Police departments are often interested in cases where tissues are already decomposed and only do bones remain to let them perform laboratory analyses. Bone is the most resistant tissue in animal body to time depending degradation and putrefaction, but it is often hard to extract DNA from it because of its highly mineralized structure, which makes DNA extraction and/or amplification hard to carry out. We have performed human nuclear DNA extraction and STR typing in three different cases, on bones and bone fragments from long time dead persons found buried, in the sea, almost completely burnt and whose tissues were already decomposed. We report these caseworks as we would like to show how forensic scientists are improving their skill in identifying people whose corps have undergone several kinds of processes, even independently on the time passed and the level of putrefaction of their tissues.