Forensic Biology Screening Workshop Other Body Fluids and Tissues

Vaginal Secretions

Vaginal secretions are a complex mixture of cells and secretions There is no confirmatory test to identify vaginal secretions Several screening tests based on microscopy have been proposed.

Vaginal Secretions Vaginal epithelial cells are large, and many contain glycogen (a polysaccharide) which can be demonstrated by staining with iodine in the form of a solution or exposing to iodine vapor. The presence of glycogenated cells is variable depending on the stage of the estrous cycle Glycogenated cells can be found in other body secretions (i.e. oral, anal)

Vaginal Secretions Based on the very high glycogen content of the vagina, vaginal secretions have a higher quantity of glycogenated cells than other body fluids Lugol's solution, first made in 1829, is a solution of iodine –Named after French doctor, Lugol

Lugol’s Staining Squamous epithelial cells with high glycogen content will show a chocolate brown color in the body of the cell (cytoplasm) with a clear round to oval shaped unstained nucleus located somewhat centrally in the cytoplasm

Lugol’s Staining HOW TO MAKE: –Iodine (0.02m) 1 g –Potassium iodide (0.06m) 2 g –Distilled water 200 ml Dissolve the potassium iodide in the water, then add iodine. HOW TO STORE: –Relatively stable at room temp or 4  C

Lugol’s Staining HOW TO PERFORM: Make a thin smear of the questioned material on a glass microscope slide Fix the smear by gentle heating Cover the smear with Lugol’s iodine solution Add an equal volume of water to the smear

Lugol’s Staining HOW TO PERFORM: Place a cover slip on the stained area Allow to stand 3-5 minutes at room temperature Observe microscopically at x

Lugol’s Staining HOW TO PERFORM: Squamous epithelial cells with high glycogen content (notably vaginal and penile urethral epithelial cells) will exhibit a chocolate-brown or tan color Other epithelial cells will exhibit a yellow or gold color

Lugol’s Staining HOW TO PERFORM: A majority of epithelial cells stained chocolate brown is a positive result. Known vaginal cells should be tested as a positive control Known buccal cells should be tested as a negative control

Lugol’s Staining epithelial cells stained chocolate brown Gold/yellow stained epithelial cells

Fecal Material

Feces are food residues passed after completion of travel through the digestive system Has a characteristic odor mainly due to skatole, an organic compound that occurs naturally in feces

Fecal Material Microscopy Microscopy has been used to identify fecal material –Looking for undigested residues of food material Chemical Tests Detection of urobilinogen, a bile pigment excreted in feces, which may be detected using its fluorescent reaction to Edelman’s reagent

Test for Urobilinogen HOW TO MAKE: Solution #1: 40% Alcoholic mercuric chloride solution –Mercuric chloride 4 g –Methanol 10 ml Mix and store in stopper bottle Solution #2: 40% Alcoholic zinc chloride solution –Zinc chloride 4 g –Methanol 10 ml Mix and store in stopper bottle Solution #3: Amyl alcohol

Test for Urobilinogen HOW TO STORE: –Relatively stable at room temp or 4  C

Test for Urobilinogen HOW TO PERFORM: Extract the suspected stain and an unstained control in 3 drops of water in separate test tubes Place an equal amount of water in a third test tube for the negative control Extract a known stain in 3 drops of water Add 3 drops of 40% alcoholic mercuric chloride to each tube

Test for Urobilinogen HOW TO PERFORM: Add 3 drops of amyl alcohol to each tube. Shake to mix Centrifuge and collect the clear supernatant Examine under UV long-wave light for any fluorescence No fluorescence should be visible at this point in the analysis Add 3 drops of 40% alcoholic zinc chloride to each tube and shake

Test for Urobilinogen HOW TO PERFORM: Incubate at room temperature for minutes Examine under long-wave UV light. The appearance of a green fluorescence is considered a positive test for urobilinogen, indicative of the presence of fecal matter

Test for Urobilinogen HOW TO PERFORM: Known fecal material should be tested as a positive control Water should be tested as a negative control Note: Safety eyeglasses which absorb ultraviolet radiation must be worn when examining for fluorescence with UV light

Urine

No confirmatory tests for the presence of urine Urine stains fluorescent under ultraviolet light –This can be useful for locating stains prior to chemical testing Has a characteristic odor Contains a large amount of urea, a chemical byproduct of normal metabolic processes in the body –Identification of high levels of urea can serve as a screening test for urine in fluids or stains –Perspiration can give reactions similar to urine

Urine Contains creatinine, which is a breakdown product of creatine (an important part of muscle) –Over time, the creatine molecule gradually degrades to creatinine –Creatinine is a waste product that is excreted from the body entirely by the kidneys –Identification of creatinine can serve as a screening test for urine in fluids or stains

Urea-Litmus Paper Test Litmus Paper test for the detection of ammonia Relies on the indirect identification of urea by reacting a test sample with urease to generate ammonia from the urea. Litmus paper is used to detect the ammonia Urea + H 2 O ↔ CO 2 + 2NH 3 A known urine sample and a blank are tested as a positive and negative control. A substrate control may be tested, as needed

Urea-Litmus Paper Test Affix a strip of litmus paper to a cork or rubber stopper that will fit into the test tubes used: cut a slit in the bottom of the stopper and insert one end of the paper. The paper strip should hang down into the tube without touching the sides of the tube or becoming wet Place a cutting from a suspected urine stain in a test tube with 3-4 drops of distilled water Add a drop of urease solution (urease mixed with distilled water) to each tube

Urea-Litmus Paper Test Incubate at 37°C for 30 minutes A definite and easily observed color change to blue on the litmus paper represents a positive result

Urea-Nitrogen Tube Test Relies on the indirect identification of urea by reacting a test sample with urease to generate ammonia from the urea. Ammonia is subsequently identified by the production of a deep blue-colored solution Urea + H 2 O ↔ CO 2 + 2NH 3

Urea-Nitrogen Tube Test HOW TO MAKE: A urease buffer solution A urea-nitrogen standard solution A phenol-nitroprusside reagent An alkaline hypochlorite reagent

Urea-Nitrogen Tube Test HOW TO STORE: The urease buffer solution is not stable at 4° C. for more than a few weeks. Make fresh solution according to concentration stated on label (30 mg urea/100 ml distilled water) Other reagents-see product inserts

Urea-Nitrogen Tube Test A known urine sample and a blank are tested as a positive and negative control. A substrate control may be tested, as needed

Urea-Nitrogen Tube Test SampleSubstrateEnzyme Negative Control1 drop H 2 O0.5 ml urease Standard1 drop urea- nitrogen standard 0.5 ml urease Positive ControlAppx 1 cm 2 stain0.5 ml urease Substrate ControlAppx 1 cm 2 substrate material 0.5 ml urease Question Sample (without urease) Appx 1 cm 2 unknown stain none Question SampleAppx 1 cm 2 unknown stain 0.5 ml urease Step 1

Urea-Nitrogen Tube Test Cover tubes and allow to stand 20 minutes at room temperature Add 1.0 ml phenol-nitroprusside reagent to each tube Add 1.0 ml alkaline hypochlorite reagent to each tube Invert to mix. Read results within 5 minutes.

Urea-Nitrogen Tube Test SampleExpected Results Negative ControlNo color StandardDeep blue color Positive ControlDeep blue color Substrate ControlNo color Question Sample (without urease)No color

Urea-Nitrogen Tube Test Question Sample If stain is urine, a deep blue color should be observed which should be as intense as the urine positive control. If the color is not as intense as the urine positive control or the color in the question sample (without urease) tube is as intense as the sample tube, this constitutes an inconclusive test result If no color is observed, this is a negative result

Creatinine Jaffe Test One of the oldest tests for the detection of creatinine-1886 Creatinine forms a red compound with picric acid (Jaffe test)

Jaffe Test HOW TO MAKE: Saturated aqueous picric acid solution –1 gram picric acid in 30 ml H 2 O 5% Sodium hydroxide Glacial acetic acid

Jaffe Test HOW TO PERFORM: Cut a portion of the suspected stain and an unstained control area and place on filter paper Cut a portion of a known urine stain and place on a separate filter paper To each, add 1 drop of saturated picric acid solution followed by 1 drop of 5% NaOH

Jaffe Test An orange coloration after 15 minutes on the questioned stain and surrounding filter paper is considered a positive result. The control area should remain yellow. Addition of 2 drops of glacial acetic acid should render the orange questioned stain yellow (de- colorization), further confirming the presence of creatinine

HAIR

Hair Composed of cylindrical structures or shafts made up of tightly compacted cells that grow from follicles Diameter ranges from µm –Depends on type of hair and body region Root material can be used for nuclear DNA testing Shaft material can be used for mtDNA testing

Hair Human hairs are distinguishable from hairs of other mammals –Human hairs are generally consistent in color and pigmentation throughout the length of the hair shaft, whereas animal hairs may exhibit radical color changes in a short distance, called banding –The medulla, when present in human hairs, is amorphous in appearance, and the width is generally less than one-third the overall diameter of the hair shaft. The medulla in animal hairs is normally continuous and structured and generally occupies an area of greater than one-third the overall diameter of the hair shaft.

Hair Structure Three cell types –Outer cuticle –Central cortex –Central medulla

Hair In some instances human hairs can be classified by racial origin such as Caucasian (European origin), Negroid (African origin), and Mongoloid (Asian origin). In some instances the region of the body where a hair originated can be determined by its gross appearance and microscopic characteristics The length and color can be determined It can also be determined whether the hair was forcibly removed, damaged by burning or crushing, or artificially treated by dyeing or bleaching.

Hair The growth phase of the hair is important in determining whether the root is suitable for nuclear DNA analysis testing Growth Cycles –Anagen phase –Catagen phase –Telogen phase

Hair Anagen Phase Active hair growth Contains nucleated cells in the root and in the surrounding sheath material Generally suitable for nuclear DNA analysis

Hair Anagen Hair

Hair Catagen Phase Transitional phase after active hair growth, cell division stops Characteristic club appearance of root May be suitable for nuclear DNA analysis

Hair Catagen Hair

Hair Telogen Phase Follows transitional phase-growth ceases Shedding phase Telogen hairs without follicular tissue may not be amenable to nuclear DNA analysis because of the lack of nucleated cells –May contain sufficient mitochondrial DNA in their roots and hair shafts for analysis

Hair Telogen Hair

Hair Basic Evaluation Steps 1.Determine if the sample is a hair 2.Determine if the hair is of human origin 3.Determine if the hair has root material-suitable for nuclear DNA analysis (Characteristic of a particular growth phase ) 4.If not suitable for nuclear DNA analysis, determine if the hair is sufficient in size for mtDNA analysis (2-3cm)

Hair DNA analysis of hair is a destructive technique and results in the consumption of portions of the hair –Hair characteristics, such as color, length, shape, and texture should be noted in the case file for future reference prior to DNA analysis Notes and digital images

Hair Hair Comparisons There are instances where useable nuclear DNA will not be extracted from a hair and comparison microscopy may be the only way to provide an association of the questioned hair to known hairs –The laboratory’s procedures should address whether microscopical hair comparisons are performed prior to DNA analysis

Hair Hair Comparisons There will be instances when mtDNA may not provide adequate discrimination among individuals. In these cases, a microscopical examination might provide sufficient discrimination of the hair to associate a questioned hair to a known hair –Most laboratories performing mtDNA analysis have procedures that require microscopical hair comparisons be performed prior to mtDNA analysis –A combination of mitochondrial DNA and comparison microscopy may help to exclude or provide a stronger association than the use of either technique alone