Development of a method for the utilization of a single sample for presumptive, confirmatory and DNA analysis of blood
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In any forensic investigation it is important to consider sample preservation. Oftentimes trace quantities of biological materials are found at crime scenes. The usual practice among forensic analysts is to take one sample of a suspected biological stain for presumptive testing, another for confirmatory testing and if both these results are positive, take a third portion for DNA analysis. This works well when sufficient sample is available, however, when trace quantities of sample are present at crime scenes, sample preservation becomes of importance. Thus, this study attempts to develop a procedure where presumptive, confirmatory and DNA analysis could be carried out on a single portion of the sample. In this study four different presumptive reagents – phenolphthalein, o-tolidine, 3, 3’, 5, 5’- tetramethylbenzidine (TMB) and luminol – were used and their effects on the ABAcard® Hematrace® immunochromatographic membrane test and subsequent DNA analysis were studied. In order to develop the method for one-sample analysis, the lowest volume of blood that gave sufficient quantity of DNA was determined by extracting different volumes (20, 10, 5, 2.5 and 1.25 μL) of whole blood. Additionally, different volumes of blood mixed with ABAcard® Hematrace® buffer were extracted. From this preliminary work it was determined that 1.25 μL of whole blood yielded sufficient DNA quantity even when mixed with the ABAcard® Hematrace® buffer. Bloodstains of 1.25 μL were then prepared and the one-sample analysis was carried out. The method developed was most successful when luminol was used as the presumptive reagent. For the bloodstains treated with the other three presumptive reagents (phenolphthalein, o-tolidine and TMB), a decrease in DNA yield was detected. This decrease was attributed to the inability of the Qiagen® QIAmp® column to adsorb the DNA after exposure to the chemical reagents and to the insolubility of the bloodstain in ABAcard® Hematrace® buffer following the addition of presumptive blood test reagents. Extraction of DNA from the ABAcard® Hematrace® immunochromatographic membrane was also carried out using the Qiagen® QIAmp® DNA investigator kit; no DNA was obtained from the membranes on which 150 μL of a dilute blood sample had been applied. This suggests that either the extraction method used was not capable of extracting the minute quantities of DNA that might be present on the membrane or there were insufficient white blood cells deposited on the membrane during the testing process. Thus, a one-sample procedure was successfully developed for bloodstains treated with luminol. A loss/reduction of DNA was observed for the samples previously exposed to phenolphthalein, o-tolidine and TMB due to the incapability of the reagents to work with silicon-based extraction chemistries. Further experimentation is needed to develop a similar procedure to be used with such presumptive testing reagents. Alternatively, a procedure can be developed that utilizes two samples: one for presumptive testing and another for confirmatory and subsequent DNA analysis, since it was observed that only the presumptive reagents, and not the ABAcard® Hematrace® buffer, interfered with DNA analysis.
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