9Identification of Semen Sperm CellsSperm cell = spermatozoanHead contains DNATail (flagella) for movementAcrosome – structure at front of headContains enzymes to penetrate egg
10Identification of Semen Searching for Semen StainsMay be on clothing, skin, bedding, etc.Visual inspection; yellowish-whiteAlternative light source: look for fluorescence
11Identification of Semen Presumptive TestsTest for seminal acid phosphatase (SAP)
12Identification of Semen Seminal Acid PhosphataseAcid phosphatases are a class of enzymes found in various living tissuesMammalian liver to cauliflower stem juiceSemen contains very high levels of seminal acid phosphataseLevels high until 40; gradual decreaseLevels not relation to sperm count
13Identification of Semen Seminal Acid PhosphataseBrentamine Fast Blue Testis the test for SAPWill react with semen diluted 500x
14Identification of Semen Seminal Acid PhosphataseMethod (Brentamine Fast Blue Test):SubstrateChromogen (color changing reagent)Seminal acid phosphatase (in semen)SAP reacts with substrate.Product of this reaction reacts with the chromogen (causes color change)
15Identification of Semen Seminal Acid PhosphataseMethod for Stain (Brentamine Fast Blue Test):Moist swab or filter paper is rubbed over stainReagent containing substrate and chromogen is dropped on swab/paper
16Identification of Semen Seminal Acid PhosphataseMethod for Stain (Brentamine Fast Blue Test):Moist swab or filter paper is rubbed over stainReagent containing substrate and chromogen is dropped on swab/paperPositive reaction is a purple color
17Identification of Semen Seminal Acid PhosphataseMethod for Swab (Brentamine Fast Blue Test):Moist filter paper is rubbed over swabReagent containing substrate and chromogen is dropped on filter paperPositive reaction is a purple colorSwabs of orifices and skin are often collected in sexual assault cases.
18Identification of Semen Seminal Acid PhosphataseMethod for Large Areas (Brentamine Fast Blue):Lay large strips of filter paper over areaMark paper and item so paper can be replaced after testSpray with water and firmly pressRemove and spray paper with reagentPositive reaction is a purple color
19Identification of Semen Seminal Acid PhosphataseFresh stains will produce a strong color change reaction.Old stains may produce a weak reactionOther materials (urine, vaginal secretions, perspiration) may produce weak reactionsExperience of analyst will determine how to proceed.
20Identification of Semen Confirmatory TestsChristmas Tree Stain (of sperm cells)Prostate-Specific Antigen (PSA)
21Identification of Semen Christmas Tree StainMicroscopic examination for sperm cellsStaining helps distinguish sperm cells from epithelial cellsTwo Dyes used:Green – Stains tailsRed – Stains heads
22Identification of Semen Christmas Tree Stain (Procedure)Collect portion of stain on moist swab or take cutting of stain.Agitate swab/cutting in small tube with water.Smear water on slide.Stain with red and green dyes.
23Identification of Semen Prostate-Specific Antigen (PSA or p30)PSA is a protein produced by the prostate gland.Can confirm semen in SAP+ samples that do not contain sperm cells.Test relies on antigen (PSA) and antibody interaction
24Identification of Semen Prostate-Specific Antigen (PSA or p30)Swab or cutting agitated in waterDrop of this solution added to test wellLiquid moves through result well
25Identification of Semen Prostate-Specific Antigen (PSA or p30)Antibody is already bound to membraneVisible test line (T) means PSA is presentOther lines are controls (show test was done properly)
27Identification of Saliva What is saliva?Water, mucus, enzymes, other stuffHumans produce 1 – 1.5 L of saliva a dayAids in digestion by lubricating food and begins breakdown of starches
28Identification of Saliva How is saliva detected?Look for the presence of AmylaseEnzyme that breaks down starchFound in many body fluids; very high in salivaTwo tests:Starch-iodine testPhadebas Reagent
29Identification of Saliva Starch-Iodine TestIodine and starch turn blueAs amylase breaks down starch, color subsidesProcedure:Gel containing starch is stained blue with iodineSample is added to well in gel. If amylase (saliva) is present, the blue color begins to vanishorBeforeNegativePositive (saliva)
30Identification of Saliva Phadebas ReagentStarch linked to a colored dyePresence of Amylase releases the dyeTwo methods for using Phadebas Reagent:Press TestTube Test
31Identification of Saliva Phadebas Reagent (Press Test)Phadebas reagent (tablets dissolved in water) sprayed on filter paper, then allowed to dryPhadebas paper placed on area to be testedPaper is sprayed with water and pressed against area
32Identification of Saliva Phadebas Reagent (Press Test)Paper is left on area and watched for blue color changeCan be watched for up to 40 minutes for color change
33Identification of Saliva Phadebas Reagent (Tube Test)Small piece containing stain is placed in tubeWater and Phadebas tablet added. Heated.Centrifuged to pellet tablet remnants and piece of cloth.Amount of color in top liquid is measured by a spectrophotometer.
34Identification of Saliva Saliva Tests (summary)Since amylase is present in other body fluids, these tests do not confirm saliva.The tests are not very sensitive (low levels of saliva may not be detected)
35Identification of Saliva DNA in SalivaWith some biological stains, DNA testing is more important than identifying the stain.If a test destroys cells, it may not be done before DNA testingExample: stamps, envelope, bite marks; saliva is inferred
36Identification of Urine Searching for UrineCases of harassment, mischief, sexual assault, etc.Stains identified through visual examination (alternate light may help)Rarely used; difficult to identify as urine and difficult to get DNA profile
37Identification of Urine Identifying UrineLook for urea or creatinineThese are in other body fluids in lower concentrationsDifficult to detect; Liquid nature of urine allows chemicals to spread out and become diluted over a large area
38Identification of Urine Identifying DNA in UrineSome epithelial cells (from urinary tract lining) and other cellsIn stains, cells are spread out; typing difficultBacteria also makes typing difficult.Cells may be concentrated from liquid samples; DNA typing possible.
39Identification of Feces Identifying FecesFeces – end product after digestionUndigested food, mucosal cells, bacteriaIdentified by greenish brown color, odor, undigested foodCan test for urobilinogen, but stain must be… apparent.
40Identification of Feces DNA testing of FecesLow cell count and high bacteria content make testing difficult, but not impossible
41Identification of Vomitus No known testPossibly look at low pH, or undigested food