RESUMEN
We report on testing 100 individuals for their shedder status with the aim of demonstrating whether the process of cell staining is reproducible when testing a large number of people. A previous report using the same method was based on 11 donors and indicated that there may be a continuum of shedder types within this small sample set. In this report we also expand the time points post-handwashing to 0, 15, 30, 60, and 180â¯min. Triplicate samples were collected from both the right and left thumbs. Samples were collected by donors placing a thumb on a clean glass slide and then adding a DNA binding dye. The number of cells were recorded within three separate square millimetre areas (cells/mm2) at 220x magnification. The experiments were conducted in triplicate on three different days, giving a total of 72 thumbprints per individual. Finally, there were 3438 observed frames in the entire dataset. Of the 100 donors, 98 gave consistent and reproducible cell number deposition. There was no difference between the cells deposited by the left and right thumbs in 13 of 15 tested. Males tended to deposit more cells than females. If applying arbitrary boundary to a cell count to definitively determine shedder status, then many of the donors fell within two categories. This study based on 100 individuals strongly suggests that shedder status is a continuum phenomenon.
Asunto(s)
Pulgar , Humanos , Masculino , Femenino , Adulto , Piel/citología , Desinfección de las Manos , Reproducibilidad de los Resultados , Recuento de Células , ADN/análisis , ADN/genética , Adulto Joven , Dermatoglifia del ADN , Persona de Mediana EdadRESUMEN
Plastic bags, such as ziplock bags, have been used to transport illicit materials worldwide; however, very few studies have tried to optimize the recovery of DNA from these items. This study reports on the best combination of swabs and moistening solution for the greatest recovery of cellular material from ziplock bags. Five swabs, two different variations of Copan Diagnostics nylon 4N6FLOQSwabs, one Medical Wire rayon DRYSWAB, one IsoHelix rayon swab, and one Livingstone cotton swab, were evaluated with two moistening solutions, Triton X-100 in either distilled water or isopropanol. Fingermarks were deposited on ziplock bags and stained with Diamond™ Nucleic Acid Dye to allow visualization of the cells pre- and post-swabbing to determine the number of cells recovered. Based on cell counting data, swabs moistened with Triton X-100 in distilled water performed better than those moistened with isopropanol. Livingstone cotton swabs had the worst recovery of cellular material, while the other swabs tested had no significant difference in their respective solutions. A comparison of the best three swabs for cellular recovery yielded no differences in the DNA concentration extracted. A linear relationship was observed between the log number of cells recovered by swabbing and the DNA concentration following extraction and quantification. The process of monitoring cell collection using fluorescence microscopy on ziplock bags allowed evaluation of swabbing efficacy. Additionally, this study highlights the ability to evaluate cellular recovery independently of traditional extraction, quantification, or profiling techniques which may unequally affect samples.
Asunto(s)
2-Propanol , ADN , Microscopía Fluorescente , Octoxinol , Manejo de Especímenes , Humanos , Manejo de Especímenes/métodos , Manejo de Especímenes/instrumentación , ADN/aislamiento & purificación , ADN/análisis , Dermatoglifia , Dermatoglifia del ADN , Recuento de CélulasRESUMEN
Latent DNA can be deposited every time a person holds or touches an item. This "touch DNA" can be crucial evidence if the item is of forensic significance. Until very recently, there were no means to visualize this DNA. The advent of using a dye that binds to DNA has opened up this possibility. The application of the dye is simple to perform, and a mobile microscope allows rapid visualization of the cellular material, even in ambient light. The dye can be applied in a solution of either 75% ethanol or water. As this is a solution-based dye, the application works best on non-absorbent surfaces.DNA within cellular material, such as dead skin cells, appears as green dots under 50X magnification; zooming to 220X magnification confirms that these are cells. The location and number of these cells can be photographed allowing a record of the presence of otherwise latent DNA.This chapter details the processes involved in the detection of latent DNA using Diamond™ Nucleic Acid Dye with both control samples (that act as very effective training samples) and the staining of evidential items. By developing skills in determining cell locations, a targeted approach to crime scene collection is now possible.
Asunto(s)
Dermatoglifia del ADN , Ácidos Nucleicos , Humanos , ADN , Microscopía , TactoRESUMEN
Disposing of items of forensic relevance in bodies of water is one countermeasure offenders can use to avoid detection. The impact of immersion in water has been explored for blood, saliva, and semen; however, few studies have assessed touch DNA. Here we report on the effect of exposure to water on the persistence of touch DNA over prolonged periods of time. To evaluate the persistence of cells from touch DNA, after water exposure, three substrates and two water types were tested: plastic, metal, and ceramic, submerged into seawater or tap water. Diamond™ Nucleic Acid Dye was used to stain cells deposited by touch. Cell counts before and after water exposure were compared to investigate cell loss over time, ranging from 6 hours to 5 days. A logarithmic increase in the percent of cells lost was observed over time when the data for substrate and water type conditions were combined. Substrate type influenced the persistence of cells, with the metal substrate retaining cells longer than plastic or ceramic. The influence of water type appeared dependent on the substrate, with varied cell persistence on metal whereas plastic and ceramic recorded similar cell loss over time between water types. The ability to visualize cells after exposure to water could assist in triaging evidence within operational forensic laboratories and allow for targeted sampling. This proof-of-concept study demonstrated that greater than 50% of cells can persist on various items submerged in aqueous environments for at least 5 days, highlighting the possibility for downstream DNA testing.
Asunto(s)
Dermatoglifia del ADN , Agua , Tacto , Colorantes , ADNRESUMEN
A wide variety of items are submitted as evidence in a forensic investigation. Identifying the location of DNA on such items is central to maximizing DNA profiling success and thus the ability to link a person of interest to a particular item or crime. Recent publications describe a fluorescent staining method using Diamond™ Dye (DD) to visualize cellular material on the surface of non-porous items (e.g., glass, plastic). However, substrates of varying porosity and background color have not yet been examined. Varying porous substrates (i.e., paper bank note, stamp, cigarette, wooden matchstick, and fabric) and non-porous substrates (i.e., enamel tooth and plastic bank note) were examined for their suitability with the use of DD. To improve the visualization of cellular material on the porous substrates, we also explored two DD diluents and adjusting image contrast. The results suggest the optimal diluent depends on the absorbent nature of the substrate. For example, ethanol was sufficient for visualization on the non-porous substrates, whereas water was better for the porous substrates. While cellular material was detected on the paper bank note, tooth, and stamp, background fluorescence or autofluorescence and surface type of matchstick prevented clear visualization on this substrate. It was also determined that by adjusting the contrast of images for tooth, paper bank note and matchstick aided in the detection of cellular material. Overall, this study extends the use of DD for latent DNA detection to absorbent substrates, highlights the limitations associated with these substrate types, and suggests modifications to improve visualization on these challenging substrates.
Asunto(s)
Dermatoglifia del ADN , ADN/análisis , Colorantes Fluorescentes , Tacto , Etanol , Femenino , Medicina Legal , Humanos , Aumento de la Imagen , Masculino , Mucosa Bucal/citología , Porosidad , Saliva/química , Piel/citología , Solventes , AguaRESUMEN
Tape-lifting is a non-destructive alternative to swabbing for collection of biological materials deposited on surfaces, especially on porous substrates. While there have been a number of studies looking at the efficiency of tapes in terms of recovery and their effect on downstream processes, none has been able to visually monitor cellular material collection. We report on a comparative study of a range of tapes regarding their collection efficiency of cellular material visualised using fluorescence microscopy, their background fluorescence after staining with DD diluted with three different solvent types and inhibition of subsequent PCR using direct PCR. Three of the fourteen tapes were selected for further testing. These were brown packing tape (Packmate™), clear tape (Sellotape®), and DNA-free tape (Lovell Surgical Solutions). These did not inhibit direct STR amplification; the other eleven tapes exhibited either high background fluorescence and/or inhibition of subsequent PCR. The effectiveness of the three tapes for the collection of cellular material was tested after tape-lifting a fingermark once, twice and ten times. The amount of cellular material within fingermarks stained with Diamond™ dye (DD) was recorded using fluorescence microscopy before and after tape-liftings. The DNA-free tape (Lovell) used in many forensic laboratories gave poor recovery compared to the other two tapes. After a tape-lift, an average of 30 % of cellular material was recovered by using DNA-free tape (Lovell), contrasting with an average recovery of 59.5 % for the clear tape (Sellotape®) and 88.8 % for the brown packing tape (Packmate™). The results presented show that standard crime scene sampling tape does collect DNA but is less effective than shop-bought tapes. Full DNA profiles can be generated from all of touched fabric samples that were collected DNA using the three tapes, triaged by DD staining and amplified by direct PCR approach.
Asunto(s)
Dermatoglifia del ADN , ADN/análisis , Manejo de Especímenes/instrumentación , Femenino , Colorantes Fluorescentes , Humanos , Masculino , Repeticiones de Microsatélite , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , TextilesRESUMEN
Touch DNA is one of the most common sample types submitted for DNA profiling. There is currently no process to visualise the presence of such DNA deposited when a person makes direct contact with items of forensic relevance. This report demonstrates the effective use of Diamond Dye to bind to DNA and allow visualisation of deposited cellular material using a mini-fluorescence microscope. Volunteers made contact with a range of items typical of those submitted as part of a forensic investigation. Contact was for less than 5 s and occurred either 15 min after hands were washed to remove any traces of DNA, and therefore under controlled conditions, or at an undefined time post handwashing to mimic real-world scenarios. Diamond Dye bound to cellular material on all the items used and in all cases it was clear where the volunteers had made this brief contact. It was also clear where no contact had been made. DNA profiling was performed on a sub-set of samples to confirm that the cellular material viewed under the microscope was human in origin and deposited by the person contacting the item; this was the result obtained in every sample tested. Diamond dye is relatively inexpensive, simple to apply, binds to the DNA in 3 s or less, has no mutagenic effects at the concentrations used, does not affect subsequent DNA profiling, and does not bind effectively to bacterial DNA. In combination with a mini-fluorescence microscope, this proof-of-concept study shows that otherwise invisible DNA deposited by touch can be visualised. The position and amount of cellular material deposited during even brief contact can be recorded allowing targeted sampling in any further DNA typing of forensically-significant items.