Disposition of Toxic Drugs and Chemicals - Lab Report Example

Disposition of Toxic Drugs and Chemicals Abstract Investigation of the body fluid is of paramount importance to support investigations of the complaint(s) or assault(s). Various laboratory methods are available today to enable forensic scientists to detect and identify extremely small quantities of body fluids. These samples incorporate materials transferred during the assault, such as hair, fibers and body fluids, particularly seminal fluid and saliva. The presence of semen in the sample indicates the prevalence of sexual assault crimes. The presumptive test used in this experiment to investigate the presence of semen in the sample was Kaye’s Test (Acid Phosphatase Test). The specific activity of L-tartrate-inhibitable acid Phosphatase (ACP). The motive of this experiment was to determine and analyze whether a particular sample was semen using the combined reagents and to exclude false positives. The set of reagents that were used helped determine the color change that occurs in a sample. Change in the color is implies the sample to be rated positive, negative or unconvincing. Keywords: Forensic Science, ACP, Kaye’s Test, Blood, Serology, DNA, Presumptive test. Introduction Forensic Science deals with the study of serology and DNA especially in the forensic crime laboratory, “serology analysis” refers to the screening of evidence for bodily fluids (Peace, 2002). It is imperative to perform body fluid detection on evidentiary items before they are sent for DNA analysis. The specimen ranges from materials, such as hairs, fibers and body fluids, particularly seminal fluid and saliva from one person to another. In the cases of sexual assault the identification of semen is imperative. For the semen identification suspect’s bodily fluid on a complainant’s body or a complainant’s bodily fluid present on clothing or items belonging to a suspect are the sample objects embracing evidentiary or probative value. It is manifested that semen found on swabs in a sexual assault kit is vital as semen can only survive inside a victim for a finite amount of time while semen stains on clothing or bedding are known to have longer duration (Gefrides, L) . There are cases where semen may not be alleged, and therefore examination of such items for its presence may not provide the evidence. It is imperative that the time between the assault and the examination can be a critical factor in the accurate diagnosis of bodily fluids because longer the time span more will be the loss of the evidence (Gefrides, L). The evidence is processed and note taking is performed during serology analysis as this is the first time the evidence is unwrapped in the laboratory. It is the responsibility of the serologists for documenting the type, quality and packaging of the evidence that is received in the laboratory. The serological methods adopted for the Forensic serology examination are straightforward. The identification of biological fluids is performed using presumptive and confirmatory testing. The presumptive encompass sensitive and specific test of the bodily fluid. It narrows the areas to be focused on. It provides the possibility of presence of bodily fluid in the specimen. This may result in false positives and therefore confirmatory tests are performed specific to the bodily fluid (Gefrides, L). Semen is a bodily fluid produced by male individual for fertilization. In the forensic study the semen is simplified in two components: seminal fluid and spermatozoa. Seminal fluid is rich in amino acids, sugars, salts, ions and other organic and inorganic materials elaborated as a heterogeneous gelatinous mass contributed by seminal vesicles, the prostate gland and Cowper’s glands. Spermatozoa refors to “sperms”, the male gametes or sex cells produced in the testis. It is evident that all men do not produce spermatozoa, especially those who have had a vasectomy, birth defects or as a result of some diseases, the seminal fluid will either not contain spermatozoa or contain very few. It is therefore imperative to forensically examine the presence of seminal fluid and the spermatozoa (Baselt, 1995). The presumptive test for the detection of seminal fluid encompass identification of an enzyme known as acid Phosphatase (AP). AP is present in the seminal fluid and originates in the prostate. Body fluids like blood, saliva, urine, vaginal secretions and others also contain AP, but the quantity of AP present in seminal fluid is higher as compared to its presence in other body tissues and therefore it is used for the detection of the presence of semen. As other body fluids also depict the presence of AP, there are chances of getting false positives. The test is performed by using Brentamine spot test. The chemicals used are α-napthyl phosphate and Diazo blue dye in a buffered solution (Gefrides, L). The positive test that detects the presence of seminal fluid changes the color to purple, because of the contamination and also because of the chemicals used in AP detection chances of false positives enhances and therefore small portion of the stain is either cut from the item or part of the stain is transferred to sterile filter paper or a sterile cotton swab for testing. The pace and also the intensity of changes in color is an indicator for the confirmation of seminal fluid. The slower pace of color change is an indication of small quantity of seminal fluid or indicates the presence of other body fluid, no color change indicate the absence of seminal fluid (Gefrides, L). It is evident that even the negative sample depicts the presence of spermatozoa and therefore further testing is essential for the confirmation of spermatozoa or the semen (Baselt, 1995). Large items are difficult to test with AP using Brentamine test, because of its time consuming and expensive procedure, moreover not all semen stains are visible to naked eyes therefore alternate light source is applied to prescreen evidence (Peace, 2002). Semen stains have the tendency to fluoresce with greater intensity than most other body fluids and therefore only the fluorescing areas if an item can be identified and used for AP testing (Gefrides, L). Further confirmation is done with the microscopic examination of the spermatozoa or the chemical detection of semen specific protein. Positive swabs or the cutting from the stain can be smeared on the slide and examined. Stains used to examine the semen are Nuclear Fast Red (red stain) and Picoindigocarmine (green stain) and are called as “Christmas Tree” stain. The heads of small spermatozoa turns red with a lighter or white tip while the tail turns blue green. This is an absolute indicator and confirms the presence of semen in the sample (Gefrides, L). Confirmatory test is done with the protein test of the semen. This is performed to confirm the presence of spermatozoa. In case of vasectomized males or males with some congenital defect of the male reproductive system, spermatozoa are not present in the semen and therefore presence of protein, prostate-specific antigen (PSA), specific to semen is tested. This protein is also referred as “p30” in forensic terminology. This is done with Ouchterlony double diffusion method or by crossover electrophoresis. Formation of band confirms the presence of protein. One step ABA card p30 test gives a pink band. Absence of band in both these cases confirms the absence of semen. Methods Drop of dd H2O was applied to 11 cotton swabs that were rubbed against 11 samples. These cotton swabs were cut into small pieces and added into labeled tubes. These 11 samples were Positive Ctrl (Neat Seminal Fluid), Negative control (water), 1:5 dilution of seminal fluid, 1:20 dilution of seminal fluid, 1:50 dilution of seminal fluid, Mayonnaise, 1:2 dilution urine, Neat saliva, 1:5 seminal fluid/bleach, Neat vaginal fluid. First of all the tubes were arranged with positive and negative controls at the two ends and then 1-2 drops of sodium α- naphthyl acid phosphate solution was added to neat seminal fluid and also to the negative control. The tubes were allowed to stand for 15-30 seconds. Then 1-2 drops of dye solution was added and the tubes were flick-mixed. The results were recorded in terms of color change and also the speed of color change. Interpretation of Results: Positive Reaction = Purple color within 10 to 15 seconds OR = Orange/red color with naphthanil diazo red within 10 to 15 seconds Negative Reaction = No color development, slight/very slow color development Inconclusive Reaction = Slow moderate to strong color development other than the color Expected (bubble-gum pink). The process was repeated with 9 other samples and the results presented in Table 1. After the completion of Kaye’s Test (Acid Phosphatase Test), ABA PSA Card Identification of P30 was performed. For each sample 150 µl of stain extract was added to the sample well “S” and after 10 minutes the results were recorded. The samples were examined for P30 test and the results are presented in Table 2. Interpretation of Results Two lines, one in the test “T” area and one in the control “C” area indicates a positive result. One line in the control “C” area indicates a negative result. If the control fails and no line appears in the control “C” area, the test result is inconclusive. Positive results may be visible as early as 2 minutes from the start of the test, depending on the concentration of human hemoglobin that is present. For negative results, however, it is important to wait a full 10 minutes. The samples were then examined microscopically and microscopic view is presented in fig. 1. Discussion The presumptive tests that were used in the experiment help investigators to detect the presence of semen at a crime scene. The identification of semen can aid in including and excluding samples from being tested in a case. In the experiment the samples produced positive and negative results. The samples were also observed microscopically to further confirm the presence of spermatozoa. This is done to eliminate the chances of reporting false positives. The samples were also tested for the presence of P30 protein. This is done as the spermatozoa cannot be seen in case of the semen of vasectomized male individual, but presence of P30 protein confirms the presence of seminal fluid to provide the confirmation of the test being performed. These test are performed in a forensic laboratory against the crime suspects and therefore confirmation of the sample is imperative as it can change the course of life. References 1. Baselt, R.,C, Cravey, R.,H. Disposition of Toxic Drugs and Chemicals in Man, 4th ed. Foster City, CA:Chemical Toxicology Institute, 1995, pp 389-391. 2. Gefrides, L. A., and Welch, K, A, Chapter 1, Serology and DNA, The Forensic Laboratory Handbook Procedures and Practice. 3. Peace, M.,R, Tarnai L.,D, Poklis A. Performance evaluation of 4 on-site drug testing devices for detection of drugs of abuse in urine. J Forensic Sci 2002; 30(1):114-122. 4. Website: STRBase – Published STR Multiplexes. April 14, 2004. Table 1 Acid Phosphatase results Sample Results Neat Seminal fluid (Positive Control) Immediate Purple color (+) Blank (water), (Negative Control) No color (-) 1:5 dilution seminal fluid Not immediate purple color (+) 1:20 dilution seminal fluid Immediate purple color in 2 seconds (+) 1:50 dilution seminal fluid 5 seconds slightly purple color change (+) 1:100 dilution seminal fluid 10 seconds no color change (-) Mayonnaise 10 seconds no color change (-) 1:2 dilution urine 10 seconds no color change (-) Neat saliva 10 seconds no color change (-) 1:5 seminal fluid/ bleach 10 seconds purple color change (+) Neat vaginal fluid Immediate purple color change (+) Table 2 P30 Results: Sample Results Neat Seminal fluid (Positive Control) (+) Blank (water), (Negative Control) (-) 1:5 dilution seminal fluid (+) 1:20 dilution seminal fluid (+) 1:50 dilution seminal fluid (+) 1:100 dilution seminal fluid (-) Mayonnaise (-) 1:2 dilution urine (-) Neat saliva (-) 1:5 seminal fluid/ bleach (+) Neat vaginal fluid (-) Fig. 1 Semen Sample Microscopic Exam Drawing APPENDIX A – REAGENTS Acid Phosphatase (AP) Buffer: • 2.5 ml Glacial acetic acid • 10.0 g Sodium acetate (anhydrous) • 450.0 ml Distilled water Mix the above components until thoroughly dissolved. The AP Buffer is stable at room temperature. α-Naphthyl Acid Phosphate Solution: • Approximately 3-4 mg of sodium α-naphthyl acid phosphate • Approximately 3 ml of Acid Phosphatase buffer Mix the above components with a plastic pipette in an appropriately labeled 10 X 75 mm test tube. Discard the solution at the end of the lab. Dye Solution: • Approximately 4 mg of o-dianisidine or naphthanil diazo red A • Approximately 3ml of Acid Phosphatase buffer Mix the above components with a plastic pipette in an appropriately labeled 10 X 75 mm test tube. Discard the solution at the end of the lab. Kernechtrot Solution (KS): • In a beaker dissolve 5 g of aluminum sulfate in 100 ml of hot distilled water. • Immediately add 0.1 g of Nuclear Fast Red and stir with a glass rod. Allow to cool and filter. The solution is stable at room temperature for up to 6 months. Picroindigocarmine Solution (PICS): • Dissolve 1 g of Indigocarmine dye in 300 ml of a commercially purchased saturated solution of picric acid. Filter and store. The solution is stable at room temperature for up to 6 months Read More