Biological Evidence: Goals 1. Identify a stain/tissue 2. Identify species of origin - is it human? 3. Individualize it i.e. get a DNA profile? 4. Compare it to a known sample
Serology and DNA Profiling Serology identification of body fluids Blood, semen, saliva, urine, feces. DNA profiling genetic identification
Serology is that stain a body fluid? Presumptive Tests could that stain be a body fluid? Easy, quick, on-site tests Low detection limits (i.e. you have to have a lot of what you are testing for) Not very specific (can get false positives)
Serology is that stain a body fluid? Confirmatory Tests is that stain a body fluid? Done in the lab (takes longer) Very specific High detection limit (i.e. do not have to have much of what you are testing for)
Blood how do we know it s blood?
Components of Blood Cellular Components Erythrocytes (Red Blood Cells) Leukocytes (White Blood Cells) Platelets Fluid (plasma) Water Salts Proteins (albumin and antibodies)
Presumptive Tests for Blood Based on blood s peroxidase activity Peroxidase an enzyme that catalyzes the reduction of hydrogen peroxide into water and oxygen The oxygen then reacts with a chemical to produce a color of give off light H 2 O 2 Heme H 2 O + [O]
Presumptive Tests for Blood Luminol Tetra-methylbenzidine Test (TMB) Reduced phenolphthalein (Kastle-Meyer) Test Leuco-crystal violet
Luminol Fluoresces if blood is present
Tetramethylbenzidine Test (TMB) 1. Wet a swab and rub on stain 2. Add 1-2 drops of benzidine reagent 3. If there is an immediate color change false positive 4. If no color- add a drop of hydrogen peroxide 5. If test is positive a blue-green color will appear
Reduced Phenolphthalein Test (Kastle-Meyer) Turns pink if positive for blood
Leuco-crystal Violet Test Turns dark purple in the presence of blood
Remember these are presumptive tests Sources of false positives Certain metals (corroded copper or nickel) Oxidizing agents (bleach) Plant peroxidases
Controls Help determine if your test is valid i.e. reagents are working, you did it properly Positive control are the reagents working? Negative control are the reagents contaminated? Substrate control does the substrate (e.g. sheet, carpet, etc.) give the expected result?
Biological Properties of Blood Main elements used in forensic labs: Red blood cells Serum proteins Red blood cells have structures called antigens on their surfaces. They are grouped into classification systems determined by their relationship to one another (blood type). Copyright 2013 Crosscutting Concepts, LLC. All Rights Reserved. www.crosscuttingconcepts.com
Biological Properties of Blood Serum proteins such as antibodies are used to test the sample to confirm that it is blood. An antibody activates or destroys a specific antigen which allows for particular reactions to occur when specific groups of antigens and antibodies are mixed. These reactions allow for determination of blood type. Copyright 2013 Crosscutting Concepts, LLC. All Rights Reserved. www.crosscuttingconcepts.com
Biological Properties of Blood The ABO group system for blood typing was first used in the early 1970 s to link blood to an individual. An individual that is type A has A antigens on their red blood cells. Type B has B antigens on their red blood cells. Type AB has both A and B antigens. Type O has neither A or B antigens. Copyright 2013 Crosscutting Concepts, LLC. All Rights Reserved. www.crosscuttingconcepts.com
Biological Properties of Blood In addition to the A and B antigens there is also another antigen, the Rh factor. Its presence is indicated by a positive sign and its absence is indicated by a negative sign. This is appended after the A/B/O indicator. Copyright 2013 Crosscutting Concepts, LLC. All Rights Reserved. www.crosscuttingconcepts.com
Blood Type Frequency Type African American Asian Caucasian Hispanic O+ 47% 39% 37% 53% O- 4% 1% 8% 4% A+ 24% 27% 33% 29% A- 2% 0.5% 7% 2% B+ 18% 25% 9% 9% B- 1% 0.4% 2% 1% AB+ 4% 7% 3% 2% AB- 0.3% 0.1% 1% 0.2% Copyright 2013 Crosscutting Concepts, LLC. All Rights Reserved. www.crosscuttingconcepts.com
Confirmatory Tests - Blood Identification Based on the presence of hemoglobin It is a protein found in red blood cells Red blood cells are anucleated (they do not have a nucleus) and are biconcave disks
Confirmatory Tests for Blood Microscope tests Takayama test Teichmann test
Takayama Test Microscopic test for hemoglobin Red/pink leaf-like crystals are formed
Teichmann Test Red/brown crystals form in the presence of blood
Confirmatory Tests for Blood Immunological Tests Ouchterlony ABAcard - Hematrace
Blood Origin Testing The Ouchterlony test will determine whether the stain is of human or animal origin. The test uses an antigen that is designed to destroy human blood. This test takes advantage of the fact that antigens and antibodies naturally move toward each other on a gel plate. Copyright 2013 Crosscutting Concepts, LLC. All Rights Reserved. www.crosscuttingconcepts.com
Ouchterlony (Diffusion) test The extracted blood stain (antigen) and the human antiserum (antibody) are placed in separate holes opposite each other on the gel. If the blood is human, a line of precipitation forms where the antigens and antibodies meet.
ABAcard - Hematrace Sample (antigen) is placed in sample well Antibodies are found on the test strip If the antigen and antibody agglutinate (clump) you get a pink line on the test strip indicated the sample contains blood!! There is also a positive control that should produce a line if the test worked properly
Next Steps 1. Read the investigation background information 2. Work on the pre-lab questions