1. Blood and Bloodstains

2. Introduction to 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. Blood Facts
The average adult has about FIVE liters of blood inside of their body, which makes up 7-8% of their body weight.
Blood is living tissue that carries oxygen and nutrients to all parts of the body, and carries carbon dioxide and other waste products back to the lungs, kidneys and liver for disposal. It also fights against infection and helps heal wounds, so we can stay healthy.
There are about one billion red blood cells in two to three drops of blood. For every 600 red blood cells, there are about 40 platelets and one white cell.

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

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

8. 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

9. Hemoglobin picks up and drops off oxygen
Introduction to Blood
ABO blood groups found on outside of cell
Hemoglobin picks up and drops off oxygen

10. Unknown Stain at a Scene
Questions to be answered:
Is it blood?
Is it human blood?
Whose is it?
Determine blood type, alcohol content, drugs present
Determine the method(s) in which blood may have been deposited

11. 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?)

12. 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?)

13. Presumptive Screening Tests
Negative result means the questioned stain is not likely blood
Positive result means the questioned stain is likely blood
Presumptive tests produce a color reaction or release of light
Tests rely on catalytic properties of blood (hemoglobin presence)
Three Categories
Color
Luminescent
Fluorescent

14. 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.

15. 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.

16. Colorless substrate + 2[OH-]  Colored product + 2H2O
Screening Tests
Based on the “peroxidase-like” activity of the blood
Linked to the hemoglobin’s heme portion
Basic reaction:
H2O2 + Heme  2[OH-]
Colorless substrate + 2[OH-]  Colored product + 2H2O

17. Screening Tests Extremely Sensitive
Presence of blood detectable even when there is no visible staining
EX: phenolphthalein – can detect 1 part blood in 1-10 million parts of dilutent
Kastle-Meyer Color Test Mixture of phenolphthalein and hydrogen peroxide on blood cause the mixture to turn pink
Because hemoglobin reacts like a peroxidase (enzyme that breaks down hydrogen peroxide), reaction will give off oxygen in presence of hydrogen peroxide

18. Hematest® Tablet
reacts with the heme group in blood causing a blue-green color

19. Look for quick blue color
TMB
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

20. Look for quick blue color
TMB
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

21. 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

22. 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
Destroys important markers for DNA fingerprinting

23. 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

24. 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?)

25. 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
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

26. 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?)

27. 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

28. Microscopic Views Bird Blood Fish Blood Horse Blood Cat Blood
Dog Blood
Fish Blood
Frog Blood
Snake Blood
Microscopic Views
Human Blood
Horse Blood

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

30. 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

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

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

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

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

35. Anti-human test Human blood is injected into an animal
Usually a rabbit
Animal will produce antibodies against human blood
Serologist will extract antibodies from the rabbit’s serum
Called Human anti-serum

36. Anti – Human Test
Layer an extract of the blood stain on top of the human anti-serum in a capillary tube
If blood is human, there will be a reaction between the human anti-serum and the unknown blood and a precipitate will form

37. Precipitin test
An antibody that reacts with its corresponding antigen will be used on the unknown stain to see if it will precipitate (solidify or crystallize)

38. False positives Contamination of the blood sample
Blood is from higher order apes
Close relatives of humans
Orangutan
Chimpanzee
Gorilla

39. Other origin tests Gel diffusion Electrophoretic method
Same basic technique as a precipitin test except it is done in a gel plate
Antigens and antibodies will be attracted towards each other
Electrophoretic method
Apply an electrical charge to accomplish same results

40. Secretor
An individual who secretes his or her blood- type antigen in body fluids
Found saliva, semen, vaginal secretions, gastric juices
Approximately 85% of people are secretors

41. 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?)

42. 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

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

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

45. Blood Terminology
ABO blood groups based on having an A, B, both or no antigens on red blood cells
Rh factor may be present on red blood cells; positive if present and negative if not
Antigen  a substance that can stimulate the body to make antibodies. Certain antigens (proteins) found in the plasma of the red blood cell’s membrane account for blood type.
Antibody  a substance that reacts with an antigen
Agglutination  clumping of red blood cells; will result if blood types with different antigens are mixed

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

47. 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

48. Genetics of Blood Types
Your blood type is established before you are BORN, by specific GENES inherited from your parents.
You inherit one gene from your MOTHER and one from your FATHER.
These genes determine your blood type by causing proteins called AGGLUTINOGENS to exist on the surface of all of your red blood cells.

49. AA or AO = Type A BB or BO = Type B OO = Type O AB = Type AB
What are blood types?
There are 3 alleles or genes for blood type: A, B, & O. Since we have 2 genes, there are 6 possible combinations.
Blood Types
AA or AO = Type A BB or BO = Type B OO = Type O AB = Type AB

50. 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

51. Blood Transfusions O A B AB
A blood transfusion is a procedure in which blood is given to a patient through an intravenous (IV) line in one of the blood vessels. Blood transfusions are done to replace blood lost during surgery or a serious injury. A transfusion also may be done if a person’s body can't make blood properly because of an illness.
Who can give you blood?
People with TYPE O blood are called Universal Donors, because they can give blood to any blood type.
People with TYPE AB blood are called Universal Recipients, because they can receive any blood type.
Rh +  Can receive + or -
Rh -  Can only receive - A B O AB
Universal Donor
Universal Recipient

52. Rh Factors A+ A- B+ B- AB+ AB- O+ O-
Scientists sometimes study Rhesus monkeys to learn more about the human anatomy because there are certain similarities between the two species. While studying Rhesus monkeys, a certain blood protein was discovered. This protein is also present in the blood of some people. Other people, however, do not have the protein.
The presence of the protein, or lack of it, is referred to as the Rh (for Rhesus) factor.
If your blood does contain the protein, your blood is said to be Rh positive (Rh+). If your blood does not contain the protein, your blood is said to be Rh negative (Rh-).
A+ A- B+ B- AB+ AB- O+ O-

53. Blood Evidence
Blood samples – Can be analyzed to determine blood type and DNA, which can be matched to possible suspects.
Blood droplets – Can be analyzed to give clues to the location of a crime, movement of a victim, and type of weapon.
Blood spatter – Can be analyzed to determine patterns that give investigators clues to how a crime might have happened.

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

55. 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

56. 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

57. 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

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
Anti-A 10 AB
A & B
none 5 O H
Anti-A & Anti-B 45

59. How common is your blood type?
46.1%
38.8%
11.1%
3.9%

60. Blood Group Markers Testing is similar to species test
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

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

62. 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

63. 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. -

64. Protein or Enzyme Markers

65. 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