Subarachnoid anesthesia vs monitored anesthesia care for outpatient unilateral inguinal herniorrhaphy.

AIM: Inguinal herniorrhaphy is one of the most commonly performed surgical procedures in a same-day surgery setting. The prerequisite of having to discharge the surgical outpatient on the same day has an influence on the choice of the anesthetic technique. METHODS: A randomized clinical trial was performed on 100 outpatients; 50 patients were enrolled in the subarachnoid anesthesia (SA) group and 50 patients in the monitored anesthesia care (MAC) group. Patients in the MAC group received local anesthesia plus target-controlled infusion propofol (LA+TCI). SA was performed using 7.5 mg 0.5% hyperbaric bupivacaine according to the selective technique. In the LA+TCI group, LA was performed with 20 mL 1% mepivacaine + 10 mL 1% ropivacaine; IV propofol sedation using TCI according to Schnider was used to obtain a Ramsay scale response of 4-5. Transferability from the operating room was evaluated based on an Aldrete score > or =9; ability to discharge from the health facility was evaluated based on a Post-Anesthesia Discharge Scoring System (PADSS) > or =9. RESULTS: Of the 100 total patients enrolled, five drop-outs were recorded in the SA group. By comparing the LA+TCI and SA groups, it was determined that the time to an Aldrete > or =9 score from the end of the procedure was 25+/-27 vs 34+/-54 min (P=0.330); the time to a PADSS 9 score was 113+/-58 vs 181+/-65 min (P<0.001); actual discharge occurred after 236+/-83 vs 289+/-78 min (P<0.01). CONCLUSIONS: LA+TCI was shown to be more effective than selective SA at low doses in terms of shorter time to recovery after unilateral inguinal herniorrhaphy procedures.

DNA extraction from blood determination membrane card test.

Among the several techniques applied in our laboratory, in order to establish human nature of blood specimens we perform an immunodiagnostic analysis through a specific membrane test card, which is usually utilized as a fast screening test for human haemoglobin in diagnostic checks. This test works with a double-step method based upon solubilization of the sample in a proper buffer and its migration on a membrane monoclonal and polyclonal antibodies specific against haemoglobin are linked to. The complexes haemoglobin-antibodies migrate through the membrane and are revealed by an immunoreactive line turning reddish-coloured to show that the reaction took place and the system works. As this test can recognize both the whole haemoglobin molecule and the degraded one, it is usually exploited in forensic sciences to detect kind and species of blood specimens related to forensic case works. However, as a consequence of the small amounts of blood in the samples collected from the crime scene we often cannot perform this kind of analysis in order to save the sample and be able to genotype it. Then, we investigated the possibility to purify DNA from the blood specimens already dissolved in the extraction buffer and perform STR typing on it with several methods and after different storage conditions and time.

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.

Experimental procedures comparing the activity of different Taq polymerases.

Forensic investigations involve several scientific branches among which biological analyses are much more frequently requested as a consequence of their importance and great versatility towards most of the traces found on the crime scene. Biological analyses are lead in subsequent steps: extraction, amplification and STR typing of the specimens collected on the crime scene. All of these techniques have been modified from original protocols according to the kind of sample to process. A critical point in our analysis is trying to amplify small amounts of DNA extracted from decomposed tissues or objects, small biological traces have been left on, with high fidelity and account. That's why we have decided to settle on an experimental procedure aimed to find the best DNA polymerase according to our purposes. We have tested different Taq polymerases on the same known DNA sample at several dilutions and have compared quality and amount of amplified DNA in order to appreciate the amplifying capability of each enzyme. These data have been analyzed as a function of the technical properties of each engineered Taq polymerase and results are shown in details.

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.

A study of case dynamic by fingerprints' fragments analysis.

Fingerprints are an extremely helpful tool for investigators as they can lead to personal identification through the observation and comparison of the characteristic minimal elements present in their structure. As forensic scientists and not only like investigators we have considered the hypothesis they may also be useful to study the dynamic of a gun shot murder in order to realize how the event took place. We have detected the presence of some papillary crest impressions on a gun which had shot and killed a person: afterwards, we have studied how those traces were oriented on the gun itself in order to determine how it was grasped, which led us to understand the murderer's behaviour on the crime scene.

A novel approach to identify the presence of fingerprints on wet surfaces.

In forensic science, one of the major problems is trying to reveal the presence of fingerprints on wet surfaces. Not often are fingerprints left in protect environments, so we have to detect their presence on the most different surfaces, after they underwent to the action of atmospheric agents or have been found, for example, on objects floating in the water. Small Particle Reagents (SPR) is a technique performed to detect latent fingerprints left on wet or moist surfaces based upon the reaction between the fatty-acid residuals present in the traces and hydrophobic tails of the specific reagents. Those tails are linked to a hydrophilic head reacting with a titanium dioxide salt giving a white precipitate plainly detectable. In this report, we want to show that exalting fingerprints left on plastic, glass and metal wet surfaces is possible with the SPR technique independently on the time fingerprints were in contact with water, as we performed in our experimental procedure. Results in details.

Cells of the human myelomonocytic line RC-2A synthesize tissue factor-like procoagulant and urokinase-type plasminogen activator.

Cells of the myelomonocytic leukemia cell line RC-2A were studied for their ability to synthesize clotting-promoting and fibrinolytic factors. The cells were observed to generate procoagulant activity (PCA) in readily measurable quantities. Incubation of RC-2A cells with phorbol myristate acetate (PMA; 3 ng/ml) or phytohemagglutinin (PHA, 10 micrograms/ml) for 18 h resulted in a 4-5-fold increase in PCA relative to unstimulated control. The PCA of RC-2A cells was tissue factor-like in that it was dependent on factor VII but not on factors VIII or IX. RC-2A cells also produced plasminogen activator (PA). Secreted PA was approximately 70% of the PA of an identical number of human monocyte-derived macrophages; fresh isolated monocytes synthesized virtually no PA. Compared to macrophages, RC-2A cells secreted less or no PA-inhibitors. Lysates of RC-2A cells contained over three times more PA than lysed macrophages. Stimulation of the cells with lectins (PHA, concanavalin A) or PMA was followed by a modest (2-3-fold) increase in PA. Enzyme immunoassay with antibodies to urokinase (u-PA) or tissue-type PA (t-PA) identified the RC-2A plasminogen activator as being of urokinase type.