INTRODUCTION TO FORENSIC SEROLOGY

1. INTRODUCTION TO FORENSIC SEROLOGY Ulfa Camelia Indiasari

2. Serology analysis in Forensic Crime Laboratory  the screening of the evidence for bodily fluid In most cases, bodily fluid identification is performed on evidentiary items before DNA analysis is attempted The type of evidentiary items submitted usually involves sexual assault cases or potential blood evidence Gefrides, L A and Welch, K E. Serology and DNA in The Forensic Laboratory Handbook. 2006

3. Forensic Serology  identification of many bodily fluid • Presumptive testing Testing that is sensitive, fairly spesific to the bodily fluid in question and can be performed quickly • Confirmatory testing Testing spesific to bodily fluid in question and sometimes also to a particular species Gefrides, L A and Welch, K E. Serology and DNA in The Forensic Laboratory Handbook. 2006

4. Identification of Semen Important in many cases of alleged sexual assault What is semen? • Semen  seminal fluid and spermatozoa • Ejaculate volume = 2 – 6 mL • 100 – 150 million sperm cells per mL • Sperm count may be much lower • Disease • Genetics • Drugs/Alcohol • Vasectomy Gefrides, L A and Welch, K E. Serology and DNA in The Forensic Laboratory Handbook. 2006

5. Identification of Semen... • Acid Phosphatase Screening • UV light • Microscopic Identification of Spermatozoa • Protein Confirmation of Semen (P30) Presumptive Confirmatory Gefrides, L A and Welch, K E. Serology and DNA in The Forensic Laboratory Handbook. 2006

6. Identification of Blood Blood identification is central to many homicide investigation The most characteristic feature of blood is hemoglobin, and forensic tests for blood are based on detecting hemoglobin or its component • Microscopic examination of blood • Chemical examination • Crystal test -- takayama test • Catalytic test – phenophtalein (PH), tetramthylbenzidine (TMB), leuchomalachite green (LMG), orthotoludine, luminol • Spectroscopic examination • Species testing – Ouchterlony test Gefrides, L A and Welch, K E. Serology and DNA in The Forensic Laboratory Handbook. 2006

7. Identification of Saliva What is saliva? • Water, mucus, enzymes, other stuff • Humans produce 1 – 1.5 L of saliva a day • Aids in digestion by lubricating food and begins breakdown of starches The presumptive testing for saliva – the detection of amylase (an enzyme which break down the starch) Gefrides, L A and Welch, K E. Serology and DNA in The Forensic Laboratory Handbook. 2006

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9. Identification of Semen Seminal Acid Phosphatase • Acid phosphatases are a class of enzymes found in various living tissues • Mammalian liver to cauliflower stem juice • Semen contains very high levels of seminal acid phosphatase • Levels high until 40; gradual decrease • Levels not relation to sperm count

10. Identification of Semen Seminal Acid Phosphatase • Brentamine Fast Blue Test • is the test for SAP • Will react with semen diluted 500x

11. Identification of Semen Seminal Acid Phosphatase Method (Brentamine Fast Blue Test): • Substrate • Chromogen (color changing reagent) • Seminal acid phosphatase (in semen) • SAP reacts with substrate. • Product of this reaction reacts with the chromogen (causes color change)

12. Identification of Semen Seminal Acid Phosphatase Method for Stain (Brentamine Fast Blue Test): • Moist swab or filter paper is rubbed over stain • Reagent containing substrate and chromogen is dropped on swab/paper

13. Identification of Semen Seminal Acid Phosphatase Method for Stain (Brentamine Fast Blue Test): • Moist swab or filter paper is rubbed over stain • Reagent containing substrate and chromogen is dropped on swab/paper • Positive reaction is a purple color

14. Identification of Semen Seminal Acid Phosphatase Method for Swab (Brentamine Fast Blue Test): • Moist filter paper is rubbed over swab • Reagent containing substrate and chromogen is dropped on filter paper • Positive reaction is a purple color Swabs of orifices and skin are often collected in sexual assault cases.

15. Identification of Semen Seminal Acid Phosphatase Method for Large Areas (Brentamine Fast Blue): • Lay large strips of filter paper over area • Mark paper and item so paper can be replaced after test • Spray with water and firmly press • Remove and spray paper with reagent • Positive reaction is a purple color

16. Identification of Semen Seminal Acid Phosphatase • Fresh stains will produce a strong color change reaction. • Old stains may produce a weak reaction • Other materials (urine, vaginal secretions, perspiration) may produce weak reactions Experience of analyst will determine how to proceed.

17. Identification of Semen Confirmatory Tests • Christmas Tree Stain (of sperm cells) • Prostate-Specific Antigen (PSA)

18. Identification of Semen Christmas Tree Stain • Microscopic examination for sperm cells • Staining helps distinguish sperm cells from epithelial cells • Two Dyes used: • Green – Stains tails • Red – Stains heads

19. Identification 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.

20. Identification 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

21. Identification of Semen Prostate-Specific Antigen (PSA or p30) • Swab or cutting agitated in water • Drop of this solution added to test well • Liquid moves through result well

22. Identification of Semen Prostate-Specific Antigen (PSA or p30) • Antibody is already bound to membrane • Visible test line (T) means PSA is present • Other lines are controls (show test was done properly)

23. Identification of Saliva Starch-Iodine Test • Iodine and starch turn blue • As amylase breaks down starch, color subsides Procedure: • Gel containing starch is stained blue with iodine • Sample is added to well in gel. If amylase (saliva) is present, the blue color begins to vanish or Before Negative Positive (saliva)

24. Identification of Saliva Phadebas Reagent • Starch linked to a colored dye • Presence of Amylase releases the dye Two methods for using Phadebas Reagent: • Press Test • Tube Test

25. Identification of Saliva Phadebas Reagent (Press Test) • Phadebas reagent (tablets dissolved in water) sprayed on filter paper, then allowed to dry • Phadebas paper placed on area to be tested • Paper is sprayed with water and pressed against area

26. Identification of Saliva Phadebas Reagent (Press Test) • Paper is left on area and watched for blue color change • Can be watched for up to 40 minutes for color change

27. Identification of Saliva Phadebas Reagent (Tube Test) • Small piece containing stain is placed in tube • Water 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.

28. Identification 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)

29. Forensic Analysis of Blood • Visual examination of evidence Presumptive screening test (Is it blood?) Confirmation test (Seriously, is it blood?) Determine species origin (human blood?) Identify the blood (whose blood is it?)

30. Presumptive Screening Tests • Negative result means the questioned stain is not likely blood • Positive result means the questioned stain is likely blood

31. Presumptive Screening Tests • Presumptive tests produce a color reaction or release of light • Tests rely on catalytic properties of blood (hemoglobin presence)

32. Presumptive Screening Tests Color Tests • Apply chromogen (color changing chemical) • Apply oxidizing agent (hydrogen peroxide) • The catalyst of the reaction is hemoglobin Rapid color change is a positive result. This means the stain is blood.

33. Presumptive Screening Tests Color Test Method • Sample stain with clean cotton swab • Add drop of chromogen • Add drop of hydrogen peroxide

34. Presumptive Screening Tests Color Test Method • Sample stain with clean cotton swab • Add drop of chromogen • Add drop of hydrogen peroxide • Alternatively, collect stain on thread; add chromogen and hydrogen peroxide in spot plate

35. Presumptive Screening Tests Color Tests Remember, other non-blood substances might catalyze the reaction also. • Chemical Oxidants • Plant materials False Positive – A positive result given by a substance that is not blood.

36. Presumptive Screening Tests 5 Types of Color Tests • Benzidine • Phenolphthalein • O-Tolidine • Tetramethylbenzidine (TMB) • Leucomalachite Green (LMG)

37. Presumptive Screening Tests Benzidine • Positive result = blue color • Carcinogen (cancer causing) in 1974 • No longer used by sane scientists

38. Presumptive Screening Tests Phenolphthalein • Positive result = pink • Some other substance produce colors other than pink (not blood) • Still used today

39. Presumptive Screening Tests O-Tolidine • Positive result = blue • Similar to benzidine; still carcinogenic as it can be metabolized to benidine • No longer used; gradually replaced by TMB

40. Presumptive Screening Tests Tetramethylbenzidine (TMB) • Positive result = Blue-green • Most common test for blood • Rub stain with moist swab • Add TMB • Add peroxidase Look for quick blue color

41. Presumptive Screening Tests Tetramethylbenzidine (TMB) • Positive result = Blue-green • Most common test for blood • Rub stain with moist swab • Add TMB • Add peroxidase Look for quick blue color

42. Presumptive Screening Tests Leucomalachite Green (LMG) • Positive result = Green • Not as sensitive as TMB or specific as phenolphthalein

43. Presumptive Screening Tests Chemiluminescence and Fluorescence • Chemiluminescence – light is emitted as a product of the chemical reation • Fluorescence – light is emitted when a substance is exposed to a shorter wavelength of light

44. Presumptive Screening Tests Chemiluminescence and Fluorescence • More sensitive than color tests • May damage blood stain (no blood/DNA typing) • Used to locate and define areas of blood • old blood stains • cleaned floor

45. Presumptive Screening Tests Luminol • Method similar to color test • Luminol is combined with oxidant and sprayed over area thought to contain blood • Emits a blue-white to yellow green glow

46. Presumptive Screening Tests Luminol

47. Presumptive Screening Tests Fluorescein • Fluorescein is combined with oxidant and sprayed over area thought to contain blood • Fluoresces when treated with a UV light • Fluorescein includes a thickener; this makes it more effective on vertical surfaces • Study showed no interference with DNA analysis

48. Presumptive Screening Tests Fluorescein

49. Forensic Analysis of Blood • Visual examination of evidence Presumptive screening test (Is it blood?) Confirmation test (Seriously, is it blood?) Determine species origin (human blood?) Identify the blood (whose blood is it?)

50. Confirmatory Tests • Due to the possibility of false positives with the presumptive tests, confirmatory tests are necessary • Confirmatory tests involve making crystals that detect the presence of hemoglobin