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Forensic identification of uncommon body fluids.pptx
- 3. Presumptive assays Urea • DMAC Assay (colorimetric and florimetric) • Microscopic crystal assay • Urease assay • Chromatography GC-MS, Thin layer chromatography Creatinine • Colorimetric assay Jaffe test • Chromatographic assay GC-MS Uric acid • Uricase assay Confirmatory assay • Tamm-Horsfall glycoprotein • 17-ketosteriods *Other bodily fluids, such as sweat, also contain these chemical components
- 4. Urine • UV examination - Urine stains fluoresce as yellow/pale blue in UV light. • Flame test - On heating gently over a flame, the characteristic odour of urine may be detected.
- 5. para-dimethylaminocinnamaldehyde (DMAC) assay light source at 473–548 nm Limitations
- 6. Urine Microcrystal test • Urea Nitrate Crystal test - An aqueous extract of the stain when reacted with one drop of conc. Nitric acid on a slide forms hexagonal urea nitrate crystals.
- 7. Urease activity The ammonia is detected using an acid-base indicator, bromthymol blue, which exhibits a blue color. Alternatively, the ammonia can be detected by manganese and silver nitrates, which exhibit a black color.
- 8. • Creatinine test (Modified Jaffe’s test) – One drop of picric acid is added to stain followed by 5% Sodium hydroxide – Brown/orange color shows presence of creatinine.
- 9. Measuring the disappearance of the absorption of uric acid at 293 nm after treatment with uricase that catalyzes the oxidation of uric acid to 5- hydroxyisourate
- 10. Confirmatory test for Urine RSID™-Urine is a lateral flow immunochromatographic strip test designed to detect the presence of the TammHorsfall (THP) glycoprotein (sometimes called uromodulin). Tamm-Horsfall is the most abundant protein present in urine. It is secreted by the thick ascending limb of the loop of Henle, and then excreted into urine at a rate of 80-200 mg/day RSID™-Urine is specific for urine and does not crossreact with any other human bodily fluids.
- 11. Confirmatory test for Urine : 17-Ketosteroids using LC-MS metabolite of testosterone and dihydrotestosterone endogenous steroid hormone precursor metabolite of testosterone
- 12. Fecal Matter • Sodomy • assault with fecal matter • Vandalism • burglary during which the perpetrator defecated at the scene
- 13. Human feces contain undigested foodstuffs, sloughed intestinal epithelial cells, intestinal bacteria, bile pigments, electrolytes, and water.
- 14. Forensic Examination Macroscopic Examination Microscopic Examination Urobilinoid Test Fecal bacterial Identification
- 15. The normal brown color of feces primarily results from the presence of urobilinoids, which are heme catabolic by-products. The characteristic odor of feces is caused by the metabolic by- products of the intestinal bacterial flora. Indole, skatole, and hydrogen sulfide are the compounds that are responsible for the odor of feces Feces Macroscopic examination
- 16. Feces Microscopic examination • Fecal matter can be transferred from samples of clothing by scraping with a sterile stainless steel spatula. The fecal matter is then hydrated in 6% formalin solution for 1-2 days prior to microscopic examination. • A small of amount of stain scraping is mounted on a slide with a drop of Lugol’s Iodine and observed microscopically for undigested vegetative matter, muscle fibres, etc • The presence of characteristic undigested foodstuffs can indicate human feces.
- 17. Urobilinoids Tests Albumin glucuronic acid Bile Unconjugation of BG IB Oxidation by IB Oxidation by IB
- 18. Schlesinger and Edelman tests Schlesinger test Sample + saturated zinc acetate (1% zinc acetate methoxyethanol solution and 0.2% Tris) urobilinoid– zinc chelation complex (green fluorescence under UV at 507 and 514 nm) Edelman test Sample + mercuric salt solution a pink-colored compound + Zinc Salt (fluorescence under UV) sonicated for 5 min, heated at 100°C for 10 min, cooled, and centrifuged Limitation: ST vs. ET Low Species specificity
- 20. Fecal Bacterial Identification 30% of fecal microbiota comprises of Bacteroides (rod-shaped, anaerobic gram-negative bacteria that digests complex carbohydrates and other substances that cannot be digested by human enzymes.) B. uniformis is not detectable in blood, saliva, semen, urine, vaginal fluids, or on skin surfaces. considered as a specific indicator bacterium for forensic fecal identification. B. vulgatus c an also be detected in vaginal fluid samples Limitations: Species non specific DIET dependent (Bacteriods rich in saturated fats and protein rich diet )
- 21. sweat
- 22. Presumptive test: Lactate, lactic acid, urea, and single amino acids Scanning Electron Microscope EDX
- 23. Presumptive test: Lactate, lactic acid, urea, and single amino acids using Raman Microscpectroscopy
- 24. Confirmatory assay for dermcidin • a class of human antimicrobial peptides of the innate immune defense system and plays an important role in protecting epithelial barriers from infections. • expressed in eccrine sweat glands • The detection can be performed using ELISA assays utilizing antibodies specific to human dermcidin. (highly sensitive for 10,000-fold dilution). • Dermcidin is encoded by the DCD gene. Its mRNA can be detected using reverse transcription polymerase chain reaction (RT-PCR) assays
- 26. Tear Basal Tears Coats the superficial layer of eye Reflex Tears Secreted in response to external stimuli Psychic Tears Secreted due to emotional and cognitive It is made up of water, electrolytes, proteins and mucins. The ratio of each component varies at different intervals in the human body.
- 27. Tear • Tests for tears • Lactoferrin is the target molecule. • Lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa • Specific testing kits with patented tech are available for testing for Lactoferrin.
- 29. Milk • Milk (Lactation) is a nutrient-rich liquid secreted by the mammary glands (breasts) of women to nourish their offspring (child). • The chief function of milk secretion is to provide nutrition. It also provides immunity, emotional connection etc. • Milk contains nutrients, proteins and lactose. • The first milk produced after childbirth is known as colostrum and contains antibodies in addition to the above. • Human milk and animal milk differs in certain means – PUFA lipoproteins and Vitamin D are present in human milk and absent in animal milk.
- 30. Milk • Tests for milk • Lactose is a target molecule – Reagent test kits • Vitamin D, LDLs can also be looked for using biochemical analysis.
Editor's Notes
- In the colorimetric method, a portion of a stain (~1 cm2 ) is cut and is extracted with 1 mL of distilled water. The extraction is transferred onto a piece of filter paper and is allowed to dry. One drop of 0.1% DMAC solution is then added to the filter paper. DMAC reacts specifically with urea, if present, producing a pink-colored (or magenta-colored) product. DMAC does not react with creatinine, ammonia, or uric acid. The appearance of a pink color within 30 min after applying the DMAC reagent is considered a positive reaction. No color change within 30 min. Limitation: However, this method is not specific to urine, as other bodily fluids such as saliva, semen, sweat, and vaginal secretions can also give positive reactions. A diluted DMAC solution, from 0.1% to 0.05%, can maintain appropriate sensitivity to urine stains and minimize false-positive reactions caused by the detection of low levels of urea that are present in other bodily fluids.
- Tamm–Horsfall protein (THP), also known as uromodulin, is the most abundant protein in urine, and accounts for 40% of the urine proteins. THP is exclusively synthesized in the epithelial cells of Henle’s loop. Under physiological conditions, an adult excretes 20–100 mg of THP into urine daily. The biological function of THP is not fully understood. It is speculated that it prevents the body from contracting urinary tract infections and from forming renal stones.
- 17-ketosteroids are substances that form when the body breaks down male steroid sex hormones. Androsterone, is an endogenous steroid hormone, neurosteroid, and putative pheromone. It is a weak androgen with a potency that is approximately 1/7 that of testosterone. Androsterone is a metabolite of testosterone and dihydrotestosterone. Dehydroepiandrosterone, also known as androstenolone, is an endogenous steroid hormone precursor. It is one of the most abundant circulating steroids in humans. DHEA is produced in the adrenal glands, the gonads, and the brain Etiocholanolone is produced from the metabolism of testosterone.
- Indole is an aromatic heterocyclic organic compound with the formula C₈H₇N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environment and can be produced by a variety of bacteria. Skatole or 3-methylindole is an organic compound belonging to the indole family. It occurs naturally in the feces of mammals and birds and is the primary contributor to fecal odor. Hydrogen sulfide is a chemical compound with the formula H ₂S. It is a colorless chalcogen-hydride gas, and is poisonous, corrosive, and flammable, with trace amounts in ambient atmosphere having a characteristic foul odor of rotten eggs.
- The formation of urobilinoids. Aged erythrocytes are disposed in the spleen, releasing hemoglobin that is broken down to heme. The heme is converted to biliverdin and is subsequently reduced to bilirubin. The bilirubin is then released into the bloodstream where it is bound to albumin, which cannot be filtrated at the glomeruli. The bilirubin is transported through the bloodstream to the liver where it is conjugated with glucuronic acid, forming water-soluble bilirubin monoglucuronide and diglucuronide. The bilirubin glucuronides are excreted into the bile and are subsequently excreted into the small intestine. In the intestines, the glucuronic acid of the conjugated bilirubin is removed. The unconjugated bilirubin is metabolized by intestinal bacteria, forming urobilinogen. A portion of the urobilinogen is further metabolized to stercobilinogen. The urobilinogen and the stercobilinogen are oxidized by intestinal bacteria, forming urobilin and stercobilin, respectively, which are excreted into the feces.
- In the Schlesinger test, a sample is mixed with saturated zinc acetate in ethanol solution to form a urobilinoid– zinc chelation complex that emits a characteristic green fluorescence under ultraviolet light. The Edelman test is a variation of the Schlesinger test. A sample is treated with a mercuric salt solution to yield a pink-colored compound. Further treatment with a zinc salt produces fluorescence. However, less fluorescence is observed in the Edelman test than in the Schlesinger test. Inconclusive and inconsistent results are often obtained using these tests where fecal material sometimes gives no visible fluorescence. Additionally, the intensity of the fluorescence observed varies between samples. The reliability and selectivity of the tests can be increased using a spectrometric measurement of the fluorescence detection of fecal urobilinoids based on the principle of the Schlesinger test. A dry sample is treated with 1 mL of zinc acetate solution (1% zinc acetate methoxyethanol solution and 0.2% Tris). The suspension is then sonicated for 5 min, heated at 100°C for 10 min, cooled, and centrifuged. The presence of urobilinoids can be detected using excitation and emission maxima at 507 and 514 nm, respectively. The disadvantages of the Schlesinger and the Edelman tests are their low species specificity as both tests cannot distinguish between human and other mammalian fecal materials.
- The identification of bacteroides can be carried out by detecting specific DNA sequences of the rpoB gene, which encodes the β subunit of bacterial RNA polymerase Two fecal predominant bacteroides species, B. uniformis and B. vulgatus, can be detected in feces. B. uniformis is not detectable in blood, saliva, semen, urine, vaginal fluids, or on skin surfaces. Therefore, B. uniformis is considered as a specific indicator bacterium for forensic fecal identification. Sometimes, B. vulgatus can also be detected in vaginal fluid samples. Therefore, precaution should be taken in interpreting the results obtained using a B. vulgatus assay. Additionally, this method alone cannot discriminate between human and animal feces. Furthermore, fecal microbial populations can be affected by the host’s diet. Individuals who consume saturated fats and proteins, which are abundant in Western diets, have predominantly Bacteroides species in their feces. However, individuals who consume a low-fat and carbohydrate-rich diet have predominantly Prevotella species, also a genus of gram-negative bacteria, in their feces