Identification and Characterization of Blood and Bloodstains

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

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

Introduction to Blood

Introduction to Blood

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.

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

Introduction to Blood White Blood Cells = DNA in nucleus

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

Introduction to Blood ABO blood groups found on outside of cell

Introduction to Blood ABO blood groups found on outside of cell

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

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

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?)

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?)

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?)

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

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

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.

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

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

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.

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

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

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

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

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

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

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

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

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

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

Presumptive Screening Tests Luminol

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

Presumptive Screening Tests Fluorescein

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?)

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

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

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?)

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

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

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

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

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

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

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

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

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

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

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?)

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

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

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

Introduction to Blood ABO blood groups found on outside of cell

Introduction to Blood ABO blood groups found on outside of cell

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

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

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

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

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

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

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

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

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

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

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. -

Protein or Enzyme Markers

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

Case File

Case File

Case File

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

Case File