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

5. Introduction to Blood

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

14. 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?)

15. 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?)

16. 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?)

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.
Chemical Oxidants
Plant materials
False Positive – A positive result given by a substance that is not blood.

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
Rub stain with moist swab
Add TMB
Add peroxidase
Look for quick blue color

28. 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

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
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

35. Presumptive Screening Tests
Fluorescein

36. 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?)

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
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?)

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

42. Species Origin Antibody is in antiserum
Antigen (serum protein) is in blood sample
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

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

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

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

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

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

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

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

50. 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?)

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 Many blood group markers, including: ABO Markers
Lewis System
Rhesus System

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

58. Blood Group Markers ABO Markers (antigen and antibodies) A Anti-B 40 B
Blood Type
Antigen
(blood group)
Antibody
% population A
Anti-B 40 B AB O

59. Blood Group Markers ABO Markers (antigen and antibodies) A Anti-B 40 B
Blood Type
Antigen
(blood group)
Antibody
% population A
Anti-B 40 B
Anti-A 10 AB O

60. Blood Group Markers ABO Markers (antigen and antibodies) A Anti-B 40 B
Blood Type
Antigen
(blood group)
Antibody
% population A
Anti-B 40 B
Anti-A 10 AB
A & B
none 5 O

61. Blood Group Markers ABO Markers (antigen and antibodies) A Anti-B 40 B
Blood Type
Antigen
(blood group)
Antibody
% population A
Anti-B 40 B
Anti-A 10 AB
A & B
none 5 O H
Anti-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+
Ladder
Type 1
Type 2
Type 3
Charge makes proteins move through gel
Different shapes move at different rates
After several minutes, their location will tell what type they are. -

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

70. Case File

71. Case File

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

73. Case File