Blood Fluid Identification - Assignment Example

Literature Review: Blood Fluid Identification Institutional Affiliation Student’s Name Blood Fluid Identification Chemical Test for Blood Kastle-Meyer Test Kastle-Meyer Test (KM test) uses the Kastle-Meyer reagent also known as phenolphthalein. Phenolphthalein is an acid-base indicator that is mainly employed in testing patent blood stains (James, Kish & Sutton, 2005). KM test is preferred for crime scene tests for blood because it has a higher stability compared to Luecomalachite Green at room temperatures whereby I can last longer without degenerating from its reduced state. Phenolphthalein makes an alkaline solution to appear pink in colour after undergoing oxidation by peroxidase when blood is present (James, Kish & Sutton, 2005). This test is quite time sensitive since its results are only dependable before 10-15 minutes elapse after which the results are rendered unreliable as false positives are produced. The KM reagent is made up of reduced phenolphthalein inside an alkaline solution that when in the presence of hemoglobin undergoes oxidation by peroxidase (James, Kish & Sutton, 2005). KM test has a sensitivity of 1:10,000 ppm just like Luecomalachite Green and these two are considered the most specific presumptive reagents (Fisher, 2004). A comparative study conducted on blood detection presumptive reagents between phenolphthalein and benzidine indicated that plant peroxidases did not generate false positives in the case of phenolphthalein but produced false positives for benzidine (Higaki & Philip, 1976). Phenolphthalein has also been observed to react with metals, metal salts, iron sources, cleaning agents, and specific plant substances. Additionally, phenolphthaleins alongside tetramethylbenzidine and benzidine have shown positive results for blood stain of about 56 years old (James, Kish & Sutton, 2005). In the KM test, the colour of the evidence under investigation influences the results because if it is red or pinkish and it percolates the swab with that colour, the swab will eventually be coloured fuchsia whether the blood is absent or present making interpretation very challenging (Tobe, Watson & Daeid, 2007). As such, this test will be ruled out and its results ignored calling for the use of an alternative colour detection presumptive reagent in another test for blood. Leuco-malachite Green (LMG) Leuco-malachite green is another widely used test for presence of blood in a crime scene. Mainly utilized to investigate the occurrence of blood stains, its preparation involves heating it until it’s colourless in an acidic mixture. LMG is mainly a laboratory based test as opposed to KM test which is often used in crime scenes. The next step involves moistening a cotton swab with a sterilised liquid and then rubbing it on the supposed blood stain (Geberth, 2006). LMG degenerates from its reduced form with time and as it undergoes oxidation process it exhibits a green colour. It requires refrigeration and constant preparation of fresh solutions. LMG is used in a similar manner to luminal when supposed blood was removed through cleaning or to create slight blood-spattered marks on a light coloured surface or material. LMG action process is an oxidation reaction that exhibits a green colour when catalysed by heme within an acetic medium and when hydrogen peroxide is an oxidizer (James, Kish & Sutton, 2005). If a colour change is observed prior to the addition of hydrogen peroxide, the test results are rendered inconclusive as a chemical oxidant is present. LMG is very specific just like Kastle-Meyer test; however, its sensitivity is relatively lower compared to other presumptive reagents. LMG sensitivity is 1:10,000 ppm which is 10 times lower than that of luminal (James, Kish & Sutton, 2005). LMG was observed occasionally destroy the DNA in samples and when samples of blood are tainted with ascorbic acid they give false positives. LMG does not provide false positive results for rust like KM test. As such, LMG is the best test when a blood stain is found on a metallic surface. Chemical Test for Saliva Phadebas Saliva can be present in a crime scene and on evidence materials in a variety of forms such as saliva deposits, cigarettes, and drinking bottles. A catalytic test to identify saliva involves the enzymatic action of alpha-amylase (α-amylase) which breaks down polysaccharides into tinier sugar particles during the digestion process. The most popular test for saliva is the phadebas test that relies on the presence of α-amylase that is found within the saliva (Mullen, 2009). The Phadebas test uses amylopectin-procion. This test is quite simple, inexpensive, swift, and highly sensitive, although it gives false positive results for faeces, urine, and hand cream. This test uses the insoluble starch polymer complex, which is broken down by carbohydrase like alpha-amylase to produce the soluble azo-dye and exhibits a blue colour change (Willott, 1974). On the other hand, α-amylase is also found in other body fluids like semen, vaginal fluids, and sweat although in smaller quantities (Auvdel, 1986). The α-amylase found in saliva is referred to as AMY1 whereas that found in semen, vaginal fluids, pancrease, sweat, and feaces is reffred to as AMY2. Despite AMY1 being mainly found in saliva compared to all other body fluids, the two forms of α-amylase are considerably impossible to tell apart through the enzymatic action (Keating & Higgs, 1994). The two can be differentiated organoleptically with the help of olfactory or visual senses. The α-amylase levels in post-menopausal vaginal releases are almost equal to the levels in saliva. As such, the test for the action of α-amylase may only provide presumptive results since it is not only limited to saliva. Red Starch Another catalytic test used to identify saliva is the Red Starch. Starch-iodine spots the α-amylase action mainly because iodine reacts with starch generating a purple-black colour (Martin, Clayson & Scrimger, 2006). When α-amylase is present, the colour is reduced visually as the amylase breaks down the starch. This method uses starch-iodine as opposed to the amylopectin-procion red used in the Phadebas test. This method is follows the principle that salivary amylase will act on starch causing a colour change since starch bears a blue colour in the presence of iodine. This method produces false positives in the case of proteins like gamma-globulin and albumin in semen and blood also hydrolyse starch leading to a change in colour. Red-Starch paper provides false positive results for feaces, urine, lotion, hand cream, and washing powders (Virkler & Lednev, 2009). Another technique that uses an insoluble amylase complex known as Amylose Azure exhibits a blue colour when broken down by alpha-amylase. This technique has a considerably higher sensitivity because it takes a shorter period, it can sense saliva mixed with other body fluids, and can sense saliva even when diluted to a ratio of 1:1000 (Virkler & Lednev, 2009). There is also the Rapignost-Amylase test strip that is mainly used to identify amylase in urine, but can also be used to identify amylase in saliva. This technique is easy and takes about 30 minutes to hydrolyse. Chemical Test for Semen Acid Phosphotase (AP) Semen is another body fluid that often features at crime scenes. It is considered to be the strongest of the various body fluids in the view of the value of the evidence. Semen can be identified using a number of presumptive tests and confirmatory tests. The occurrence of semen can be effectively identified through the use of histological methods as opposed to saliva and blood. One popular histological method is the haemotoxylin and eosin (H&E) test that stains the nucleus of the spermatozoa blue and the cytoplasm pink (Allery, Telmon, Mieusset, Blanc & Rougé, 2001). The presence of semen can be substantiated through the microscopic discovery of a single sperm cell. Presumptive tests are used to detect semen in garments and swabs before the extraction for H&E tests (Sensabaugh, 1979). Presumptive tests that can be used to discover semen include the use of an ALS like the ultraviolet light which is an easy and non destructive technique. ALS devices used to detect semen include Blue-maxx BM500, Wood’s Lamp (WL), and Polilight. Blue-maxx Bm500 has shown 100% sensitivity to semen stains in tests with other fluids. In similar tests, Wood’s Lamp (WL) which has a wavelength ranging between 320-400 nm did not give precise results and in some instances failed to sense semen stains and provided false positive results for creams and ointments. Seminal acid phosphatase (SAP) is the most popular and widely used presumptive test for the occurrence of semen. Acid phosphatase is an enzyme with the capacity to catalyse the hydrolysis of organic phosphates to produce a substance that reacts with a diazonium salt chromogen resulting in the change of colour (James, & Nordby, 2003). This is the underlying standard for all the variations of the seminal acid phosphatase test. One accepted color producing mixture is the Brentamine Fast Blue and the alpha-naphthyl phosphate. Another mixture that has given good results is the alphanaphthol with Fast Red AL and the beta-naphthol with Fast Garnet B (Saferstein, 2002). One key reagent that has also been used is sodium thymolphthalein monophosphate because of its high stability, selectivity, and minimal hazardous risk. Another reagent used is the mixture of NaNO3, pnitroaniline, a-naphthyl phosphoric acid, and aqueous magnesium chloride. Indeed the SAP technique may not be confirmatory because there are some false positives like vaginal acid phosphatase (VAP) and specific plant substances. However, these false positives for VAP can be effectively evaded by monitoring a colour change that takes place exactly between 5-30 seconds because VAP does not usually exhibit a false positive so suddenly (Saferstein, 2002). A number of techniques have been designed to make a distinction between SAP and VAP. These techniques entail the use of acrylamide gel electrophoresis and isoelectric focusing and to separate the two acid phosphatases. Additional demerits of SAP tests are based on the fact that the enzyme loses its effectiveness due to mold, heat, chemicals, or putrefaction. Chemical Test for Protein BCA Protein Identification Assay In alkaline solutions, Cu2+ attaches to the peptide bonds of proteins. The Cu2+ can be reduced to Cu+ by Cys, Tyr, and Trp producing a modest purple colour relative to the concentration of proteins (Kit). The protein concentration is established using this color in a relatively insensitive Biuret Assay but its sensitivity can be boosted using Bicinchoninic acid (BCA) (Yalamati, Bhongir, Karra & Beedu, 2015). As the Cu+ attaches to BCA, it exhibits a strong purple colour that is comparative to the level of protein concentration. It is important to note that the colour yield corresponding to each milligram of protein is not stable for all proteins since all proteins have varying levels of reducing amino acids. The level of reducing amino acids in a protein will directly be proportional to the level of absorbance values for that protein (Yalamati, Bhongir, Karra & Beedu, 2015). Despite this difference, the BCA Assay technique is quite important when monitoring changes in the level of proteins and in particular when undertaking protein purification. Perfoming BCA Assay at temperatures of 60ºC generates a less inconsistency among proteins compared to room temperature (Yalamati, Bhongir, Karra & Beedu, 2015). The BCA Assay technique can produce errors if the buffers contain non-protein reducing substances that can reduce Cu2+ to Cu+ although this can be prevented by using buffer-only samples. The Bradford Dye Binding (Bio-Rad) Protein Identification Assay Bio-Rad Assay is a dye-binding assay made possible by the varying colour change of a dye in the presence of different concentrations of protein (Manual). When van der Waals and noncovalent electrostatic linkages with proteins take place, the absorbance limit for an acidic substance made of Coomassie Brilliant Blue G-250 dyechnages from 465nm to 595nm (nach Bradford). Because of the ionic nature of the dye, its sensitivity to proteins and particularly those with Lys, His, and Arg content is very high. This technique is gaining prominence in various applications such as research due to its simplicity, speed, capacity to form a stable coloured complex with just one step, and absence of obstructions that hinder the use of other protein assays. References Allery, J., Telmon, N., Mieusset, R., Blanc, A., & Rougé, D. (2001). Cytological Detection of Spermatozoa: Comparison of Three Staining Methods. Journal Of Forensic Sciences, 46(2), 14970J. http://dx.doi.org/10.1520/jfs14970j Auvdel, M. (1986). Amylase Levels in Semen and Saliva Stains. Journal Of Forensic Sciences, 31(2), 426-431. http://dx.doi.org/10.1520/jfs12272j Fisher, B. A. (2004). Techniques of crime scene investigation. Boca Raton, FL: CRC Press. Geberth, V. J. (2015). Practical homicide investigation: tactics, procedures, and forensic techniques. Boca Raton: CRC Press. Higaki, R., & Philp, W. (1976). A Study of the Sensitivity, Stability and Specificity of Phenolphthalein as an Indicator Test for Blood. Canadian Society Of Forensic Science Journal, 9(3), 97-102. http://dx.doi.org/10.1080/00085030.1976.10757252 James, S. H., & Nordby, J. J. (2003). Forensic science: an introduction to scientific and investigative techniques. Boca Raton, FL: CRC Press. James, S. H., Kish, P. E., & Sutton, T. P. (2005). Principles of bloodstain pattern analysis: theory and practice. Boca Raton, FL: CRC. Keating, S., & Higgs, D. (1994). The detection of amylase on swabs from sexual assault cases. Journal Of The Forensic Science Society, 34(2), 89-93. http://dx.doi.org/10.1016/s0015-7368(94)72889-9 Kit, B. P. Q. KB-03-005 200 tests (96 well plate). Manual, I. Quick Start™ Bradford Protein Assay. Martin, N., Clayson, N., & Scrimger, D. (2006). The sensitivity and specificity of Red-Starch paper for the detection of saliva. Science & Justice, 46(2), 97-105. http://dx.doi.org/10.1016/s1355-0306(06)71580-5 Mullen, C. (2009). Amylase: Phadebas test, saliva. Wiley Encyclopedia of Forensic Science. nach Bradford, M. Bio-Rad Protein Assay. Saferstein, R. (2002). Forensic science handbook. Upper Saddle River, NJ: Prentice Hall. Sensabaugh, G. (1979). The Quantitative Acid Phosphatase Test. A Statistical Analysis of Endogenous and Postcoital Acid Phosphatase Levels in the Vagina. Journal Of Forensic Sciences, 24(2), 346-365. http://dx.doi.org/10.1520/jfs10841j Tobe, S., Watson, N., & Daéid, N. (2007). Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular-Weight DNA. Journal Of Forensic Sciences, 52(1), 102-109. http://dx.doi.org/10.1111/j.1556-4029.2006.00324.x Virkler, K., & Lednev, I. K. (2009). Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Science International, 188(1), 1-17. Willott, G. (1974). An Improved Test for the Detection of Salivary Amylase in Stains. Journal Of The Forensic Science Society, 14(4), 341-344. http://dx.doi.org/10.1016/s0015- 7368(74)70923-9 Yalamati, P., Bhongir, A. V., Karra, M., & Beedu, S. R. (2015). Comparative Analysis of Urinary Total Proteins by Bicinchoninic Acid and Pyrogallol Red Molybdate Methods. Journal of clinical and diagnostic research: JCDR, 9(8), BC01-4. Read More