1. Identification and Characterization of Blood and Bloodstains

2. Summary Introduction to Blood Identification of Blood as Blood Determining Species Origin of Blood Identifying Humans using Blood

3. Introduction to Blood What you should already know: It’s that red stuff that comes out of your fingers following a kitchen accident.

4. Introduction to Blood

6. What you should already know: It’s that red stuff that comes out of your fingers following a kitchen accident. Lets look at blood.

7. Introduction to Blood Red Blood Cells = No DNA White Blood Cells = DNA in nucleus

8. Introduction to Blood White Blood Cells = DNA in nucleus

9. Introduction to Blood RBC = no DNA; WBC = yes DNA All blood cells have blood groups on outside of cell.

10. Introduction to Blood ABO blood groups found on outside of cell

11. Introduction to Blood ABO blood groups found on outside of cell

12. Introduction to Blood RBC = no DNA; WBC = yes DNA All blood cells have blood groups on outside of cell. Red Blood Cells contain the protein hemoglobin, which carries oxygen

13. Introduction to Blood Hemoglobin picks up and drops off oxygen

14. Forensic Analysis of Blood 1. Visual examination of evidence 2. Presumptive screening test (Is it blood?) 3. Confirmation test (Seriously, is it blood?) 4. Determine species origin (human blood?) 5. Identify the blood (whose blood is it?)

15. Forensic Analysis of Blood 1. Visual examination of evidence 2. Presumptive screening test (Is it blood?) 3. Confirmation test (Seriously, is it blood?) 4. Determine species origin (human blood?) 5. Identify the blood (whose blood is it?)

16. Forensic Analysis of Blood 1. Visual examination of evidence 2. Presumptive screening test (Is it blood?) 3. Confirmation test (Seriously, is it blood?) 4. Determine species origin (human blood?) 5. Identify the blood (whose blood is it?)

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

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

19. 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.

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

21. 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

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

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

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

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

26. 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

27. Presumptive Screening Tests Tetramethylbenzidine (TMB) Positive result = Blue-green Most common test for blood 1. Rub stain with moist swab 2. Add TMB 3. Add peroxidase Look for quick blue color

28. Presumptive Screening Tests Tetramethylbenzidine (TMB) Positive result = Blue-green Most common test for blood 1. Rub stain with moist swab 2. Add TMB 3. Add peroxidase Look for quick blue color

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

30. 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

31. 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

32. 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

33. Presumptive Screening Tests Luminol

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

35. Presumptive Screening Tests Fluorescein

36. Forensic Analysis of Blood 1. Visual examination of evidence 2. Presumptive screening test (Is it blood?) 3. Confirmation test (Seriously, is it blood?) 4. Determine species origin (human blood?) 5. Identify the blood (whose blood is it?)

37. 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

38. Confirmatory Tests Teichmann Test and Takayama Test Small amount of blood added to microscope slide Chemical solution is added Slide is heated to form crystals Crystals viewed under microscope

39. Forensic Analysis of Blood 1. Visual examination of evidence 2. Presumptive screening test (Is it blood?) 3. Confirmation test (Seriously, is it blood?) 4. Determine species origin (human blood?) 5. Identify the blood (whose blood is it?)

40. Species Origin Most methods test for serum proteins Serum proteins are found in all animals, but are slightly different Species ID methods based on antigen/antibody interactions

41. Species Origin Antibody = produced when foreign serum protein is detected Certain antibody will only attach to one species’ serum protein Antigen = serum protein

42. Species Origin Human antiserum will only attach to human blood sample Rabbit antiserum will only attach to rabbit blood sample Dog antiserum will only attach to dog blood sample Antibody is in antiserum Antigen (serum protein) is in blood sample

43. Species Origin Antiserum in heavy bottom layer Ring Precipitin Test Blood sample (dilute) in top layer

44. Species Origin Antiserum in heavy bottom layer Ring Precipitin Test Blood sample (dilute) in top layer Precipitate means blood and antiserum species match

45. Species Origin Ring Precipitin Test Human Antiserum Yes line = Yes human blood

46. Species Origin Ring Precipitin Test Human Antiserum No line = Not human blood

47. Species Origin Antiserum placed in center Ouchterlony Double Diffusion Several bloodstains tested at one time White line means antiserum and blood match Human antiserum Human blood Not human blood

48. Species Origin Crossed-Over Electrophoresis Antiserum and Blood move toward each other Antiserum Blood Stain Gel Holes

49. Species Origin Crossed-Over Electrophoresis Antiserum and Blood move toward each other Antiserum Blood Stain If line forms, antiserum and blood match

50. Forensic Analysis of Blood 1. Visual examination of evidence 2. Presumptive screening test (Is it blood?) 3. Confirmation test (Seriously, is it blood?) 4. Determine species origin (human blood?) 5. Identify the blood (whose blood is it?)

51. Genetic Markers in Blood If a stain is blood, and it is human blood, then whose is it? Blood Group Markers Protein/Enzyme Markers DNA

52. Blood Group Markers ABO Markers Lewis System Rhesus System Many blood group markers, including:

53. Blood Group Markers ABO Markers Look at antigens on Red Blood Cells In this case, antigens are glycoproteins and are attached to the outside of the cell

54. Introduction to Blood ABO blood groups found on outside of cell

55. Introduction to Blood ABO blood groups found on outside of cell

56. Blood Group Markers ABO Markers (antigen and antibodies) A person will have antibodies (A or B) to whatever blood group he/she doesn’t have Otherwise, a persons blood would clump up and cause death

57. Blood Group Markers ABO Markers (antigen and antibodies) Blood Type Antigen (blood group) Antibody % population A B AB O

58. Blood Group Markers ABO Markers (antigen and antibodies) Blood Type Antigen (blood group) Antibody % population AAAnti-B40 B AB O

59. Blood Group Markers ABO Markers (antigen and antibodies) Blood Type Antigen (blood group) Antibody % population AAAnti-B40 BBAnti-A10 AB O

60. Blood Group Markers ABO Markers (antigen and antibodies) Blood Type Antigen (blood group) Antibody % population AAAnti-B40 BBAnti-A10 ABA & Bnone5 O

61. Blood Group Markers ABO Markers (antigen and antibodies) Blood Type Antigen (blood group) Antibody % population AAAnti-B40 BBAnti-A10 ABA & Bnone5 OHAnti-A & Anti-B 45

62. Blood Group Markers ABO Markers Testing is similar to species test An anti-A, anti-B, or anti-AB antiserum (containing antibodies) is reacted with the blood stain to detect blood cells A, B, or AB blood cells are reacted with a blood stain to detect antibodies

63. Blood Group Markers ABO Markers Tests can get complicated with absorbing and releasing cells Final step is usually testing for agglutination (blood clumping)

64. Blood Group Markers ABO Markers A cells clump with anti-A B cells clump with anti-B AB cells clump with both O cells do not clump

65. Other Markers Protein or Enzyme Markers Some proteins or enzymes can be in different forms (different shapes) These differences can be detected by separating the proteins in a gel by electrophoresis

66. Protein or Enzyme Markers Charge makes proteins move through gel Different shapes move at different rates After several minutes, their location will tell what type they are. + - Ladder Type 1Type 2Type 3

67. Protein or Enzyme Markers

68. Genetic Markers in Blood ABO blood typing and protein analysis may help eliminate a suspect. Since there are only a small number of types (ABO = 4 types), a match does not mean the stain definitely came from a certain person DNA testing can identify a person, and is becoming just as easy as the above tests

69. Case File Ring Precipitin Test Human Antiserum Yes line = Yes human blood