Forensic Serology

1. Forensic Serology Objectives: Identify and describe the components of human blood List the A-B-O antigens and antibodies found in the blood for each of the four blood types: A, B, AB, and O. Explain why agglutination occurs. Explain how whole blood is typed. Describe tests used to characterize a stain as blood. Explain the significance of the precipitin test to forensic serology. Explain the differences between monoclonal and polyclonal antibodies. Define chromosome and gene. How is the Punnet square used to determine the genotypes and phenotypes of offspring? List the laboratory tests necessary to characterize seminal stains. Explain how suspect stains are to be properly preserved for laboratory examination. Describe the collection of physical evidence related to a rape investigation. Created by C. Ippolito January 2008

2. Blood • complex mixture of cells and fluid • Formed Elements • living component • cells suspended in the liquid • Plasma • nonliving fluid matrix of unclotted blood Created by C. Ippolito January 2008

3. Plasma • 55% of whole blood • 90% water • 10% dissolved components • nutrients • sugars, lipids (fats), amino acids • gases • CO2 (bicarbonate ion HCO3-) • salts • ionic compounds containing (Na+, K+, Mg2+, Cl-) • hormones • plasma proteins • albumen – maintains osmotic pressure, pH buffering • globulins - antibodies – protective proteins of immune response • fibrinogen – protein that contributes to blood clotting mechanism Created by C. Ippolito January 2008

4. Formed Elements • 45% of whole blood • Three main blood cell types • Erythrocytes (RBC) • transport O2 • Leukocytes (WBC) • disease defense • Thrombocytes (platelets) • clotting mechanism • cell pieces • initiate clotting Created by C. Ippolito January 2008

5. Erythrocytes • most numerous blood cells • anucleate – no nucleus • hemoglobin (Hb) • iron containing protein • transports O2 • biconcave disk • increases surface area • membrane antigens • determine blood types • Anemia • lack of RBC • Polycythemia • excess RBC Created by C. Ippolito January 2008

6. Leukocytes • less numerous blood cells • defense against • bacteria, viruses, parasites, tumor cells • Leukocytosis • high WBC count • indicative of infection • Leukopenia • low WBC count • corticosteroid use, certain drugs, anticancer agents Created by C. Ippolito January 2008

7. Types of Leukocytes • Neutrophils • phagocytes – “eating cells” • attack in acute (short-term) infections • Eosinophils • attack parasitic worms • numerous during allergies • Basophils • release histamine • vasodilator releases plasma and WBC at infection site • Lymphocytes • B-cells – release antibodies • T-cells – attack viruses and tumors, activate B-cells Created by C. Ippolito January 2008

8. Human Blood Groups • RBC antigens • antigen • identifying protein that is genetically determined • recognized by a “body” as being foreign • antibody • protein released by B-cells • “match” antigens’ shape • minimum of 2 active sites (bivalent) • Agglutination • antibodies attach to RBC antigens • RBC interconnect through antibodies • RBC clump together clogging BV • Serum • plasma with antibodies Created by C. Ippolito January 2008

9. RBC Agglutination • RBC membrane antigen • Antibody matches and attaches to multiple RBCs Created by C. Ippolito January 2008

10. ABO Blood Typing Created by C. Ippolito January 2008

11. Type AB Blood • RBC has both antigens A and B • reacts with anti-A serum • reacts with anti-B serum Created by C. Ippolito January 2008

12. Type A Blood • RBC has only antigen A • reacts with anti-A serum only Created by C. Ippolito January 2008

13. Type B Blood • RBC has only antigen A • reacts with anti-B serum only Created by C. Ippolito January 2008

14. Type O Blood • RBC has no antigen • no reaction with anti-A serum • no reaction with anti-B serum Created by C. Ippolito January 2008

15. Blood Type Distribution • In the US a “typical” ABO distribution is as follows: • 43% type O • 42% type A • 12% type B • 3% type AB • ABO Blood Type distributions Created by C. Ippolito January 2008

16. Immunoassay • Using antibody-antigen reactions to identify substances • original pregnancy tests (clotting to HCG) • use rabbits to make antibodies to react with drugs • antibodies (sera) then used in urinalysis • opiates, cannabinoids, cocaine, amphetamines, phencyclidine, barbiturates, methadone can be identified, • presumptive testing, results must be confirmed by additional tests Created by C. Ippolito January 2008

17. Radioimmunoassay (RIA) • uses drugs labeled with radioactive tags • can react with substances chemically similar to target substance • also needs confirmation Created by C. Ippolito January 2008

18. EMIT • Enzyme-Multiplied Immunoassay Technique • add antibodies to urine for particular drug • add chemically labeled version of presumed drug • competition between labeled and unlabeled • amount of labeled drug left in test related to amount of original drug in sample • labeled drug left over means original drug combined with antibodies and was removed • used to test for THC (tetrahydrocannabinol) and it metabolites such as THC-9-carboxylic acid • shows marijuana use but can yield positive results for up to 10 days after last use Created by C. Ippolito January 2008

19. Polyclonal and Monoclonal Antibodies • Polyclonal antibodies • multiple antibodies produced to attack many different parts of a single antigen • preferable in immune response against a disease organism • Monoclonal antibodies • single antibody produced to attack a specific part of a specific antigen • preferable in an “assay” test to identify one particular substance • hybridoma cells • mouse spleen cells and blood cancer cells combine producing a single antibody Created by C. Ippolito January 2008

20. Forensic Characterization of Blood Stains • Three questions must be answered: • Is it blood? • Color Tests • Luminol Test • Microcrystalline Test • Is it human blood? • Precipitin Test • Gel Diffusion Test • Can it be associated with a particular person? • ABO typing (rarely done) • DNA profiling Created by C. Ippolito January 2008

21. Color Tests • Hemoglobin – acts like peroxidase • enzyme accelerates oxidation in presence of hydrogen peroxide causing a color change • enzyme also found in potatoes and horse radish • pink color indicates presence of hemoglobin • BenzidineTest • discontinued as a carcinogen • Kastle-Meyer Test • uses phenolphthalein • Hemastix • dipstick test for blood in urine • used as presumptive blood test when moistened with distilled water • turns green when in contact with blood Created by C. Ippolito January 2008

22. Luminol Test • Luminol • luminol + hydrogen peroxide  3-aminophthalate • luminescence rxn • chemical produces blue light • does not affect subsequent DNA testing Created by C. Ippolito January 2008

23. Microcrystalline Tests • addition of chemicals causes hemoglobin to crystallize • susceptible to contaminants giving false positives • Takayama Test • Teichmann Test • Wagenhaar test Created by C. Ippolito January 2008

24. Precipitin Tests • Human antiserum • derived from animal antibodies produced in response to exposure to human blood • rabbits, cat, dogs, deer used as sources • reaction of antigen-antibody forms cloudy ring where they meet in capillary/test tube Created by C. Ippolito January 2008

25. Gel Diffusion Test • antigen-antibody reaction • coagulate when meet • very sensitive • works with dried blood stains after 15 years • can be diluted to 1/256 and still react Created by C. Ippolito January 2008