Identification and Characterization of Blood and Bloodstains

1
Identification and Characterization of Blood and
Bloodstains

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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

Its 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

Its 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

• 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

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 doesnt have

• Otherwise, a persons blood would clump up and
  cause death

57
Blood Group Markers

• ABO Markers (antigen and antibodies)

58
Blood Group Markers

• ABO Markers (antigen and antibodies)

59
Blood Group Markers

• ABO Markers (antigen and antibodies)

60
Blood Group Markers

• ABO Markers (antigen and antibodies)

61
Blood Group Markers

• ABO Markers (antigen and antibodies)

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