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Blood Forensic Serology and Blood Spatter Analysis Blood • A complex fluid that consists of two portions: – Cellular elements – Red Blood Cells, White Blood Cells and Platelets – Plasma (or serum) – liquid portion of the blood which contains dissolved substances Red Blood Cells - Erythrocytes • Small, biconcave discs with no nucleus • 5 million per cubic ml. • Carry oxygen and carbon dioxide using red pigment (hemoglobin) • Live for about 120 days, then are removed by the liver or engulfed by white blood cells White Blood Cells - Leukocytes • Larger than RBC’s and have nuclei (which may be fragmented as in this neutrophil) • Function as the immune system – destroy foreign cells • Many different types and names (eg. Monocytes, macrophages, lymphocytes, T cells,B cells, etc.) Platelets Thrombocytes • Tiny cellular fragments with no nuclei • Formed in bone marrow with WBC’s and RBC’s • Contain proteins that are released when a wound occurs to initiate the process of blood clotting Bone Marrow • Located within the large bones (especially the leg bones and pelvis) is a tissue that produces all three types of blood cells • Since RBC’s and platelets cannot reproduce, without functioning bone marrow we would soon die Plasma • The composition of whole blood varies – but if centrifuged it consists of mostly plasma • This liquid contains many important proteins (like antibodies, hormones, carrier molecules) that are essential to proper body functioning • Plasma is about 90% water, 7-9% protein and also contains glucose and other nutrients, waste products, dissolved gases etc. Blood Testing • A great deal can be learned about a person and/or their circumstances with the proper blood test • Since there are dozens of even routine tests, the key in forensics is to order the proper test • Standard tests like the CBC (complete blood cell) can reveal large changes in blood composition • Changes in blood enzyme levels can be detected when tissue (like heart) is damaged • Changes in levels of some proteins in the blood can signal cancer, or liver disease, etc. Some disorders seen with a simple blood test Sickle Cell Anemia Identification of Blood • Forensic investigators often want to know whose blood they have collected from the crime scene • Several methods have been devised, but all involve the immune system • Blood typing was first done by Karl Landsteiner in 1901 • Transfusions became safer, and some degree of identity was assigned to blood • Presently DNA fingerprinting is the gold standard for identifying blood (covered later) Blood Types • The ABO system relies on antigens – proteins on the outer membrane of RBC’s • There are only two types of these proteins in this blood system: A and B • Cells can have only A antigens(Type A), only B antigens (Type B), both A and B antigens (Type AB) and no antigens for this system (Type O) Blood Types • If a person does NOT normally produce an antigen, they can make antibodies against that foreign antigen • These antibodies will clump (agglutinate) the blood cells together and make it impossible for proper circulation to occur • In the lab, these antibody reactions are used to type blood Agglutinated vs. Normal Blood Types Blood Types • Sera are produced that contain anti-A and anti-B antibodies (horses) • These sera are applied to blood on a plate • Blood type A will agglutinate when exposed to anti-A serum, blood type B will agglutinate when exposed to blood type B serum • What will blood types AB and O do when exposed to the sera? Blood Types In North America a typical distribution of blood types • Matching one of only 4 types is less than definitive • Before DNA testing, 5 or 6 blood typing systems were used to further identify the blood Immunoassays • Many drug tests now use the immune system • Antibodies can be produced in animals (harmless) by first combining the drug with a protein and injecting it into the animal • The animal will produce antibodies to the drug • These antibodies can be removed from the animal, purified, and used in tests to detect the drug EMIT Test • A typical immunoassay for drugs • Add antibodies to a blood or urine sample • Any drug present (eg. Heroin) will bind to the antibodies • Then, enzyme labeled drug is added to the urine • The quantity of enzyme-labeled heroin left over is measured and this value is related to the concentration of the drug in the sample • Caution is needed since it is possible that the antibodies can combine with related drugs – positive results must be confirmed with other tests Blood Stains Finding Blood • In some cases, blood will be obvious at the crime scene • In others, the perpetrator may have tried to clean up the blood • Often this blood can be visualized using a number of methods The Kastle-Meyer Test • Used to use benzidine (carcinogenic), now uses phenolphthalein – turns deep pink when exposed to hemoglobin • Not specific for hemoglobin – some vegetable substances like potatoes and horseradish may test positive • Also, Hemastix® – usually used as dipsticks for urine testing – are convenient tests for trace amounts of blood Luminol • Luminol reagent is sprayed onto surface • Region is darkened, luminol glows • Can detect bloodstains diluted up to 10 000 times • does not interfere with future DNA tests Precipitin Test • Precipitin tests rely on antisera produced in much the same way as for immunoassays • Whole human blood is injected into the animal (or animal blood) • Antibodies are extracted and used to react with the blood stain to determine if it is human blood • Very sensitive – can detect bloodstains even 15 years old or older (even mummies!) • Three methods of visualizing results are used: capillary tube method, gel diffusion, and electophoretic method Capillary Tube Precipitin Test Extract of bloodstain layered on antiserum to human blood Cloudy ring between layers indicates a reaction – thus the blood is human Gel Diffusion Precipitin Test Bloodstain and human antiserum placed in different wells on an agar plate Antigen and antibody, if matched, will tend to diffuse toward each other Line of precipitation can be seen – positive test for human blood Electrophoretic method is similar – uses electricity to draw antigens and antibodies through the gel Typing of Dried Stains • Although it is possible to determine if there is blood present and if it is human when dry, typing is not possible because the RBC’s rupture • Procedures to type dry blood have been developed, but have largely been replaced now with DNA fingerprinting technology Stain Patterns of Blood Surface Texture • Surface is extremely important to the interpretation of bloodstain evidence • In general, the harder and less porous the surface, the less spatter results • Control experiments on identical (or similar) surfaces must be conducted to determine distance of spatter, direction, etc Direction of Travel • The blood’s direction of travel can be determined by the stain’s shape • The pointed end of a bloodstain always faces its direction of travel Impact Angle • The degree of circular distortion is an indicator of the angle of the trajectory of the blood before it hit the object • As the angle increases, the stain becomes more elongated Effect of Gravity The position of an object when blood hit it can be determined by the effect of gravity on the blood drop after it contacts the surface Here, the effect of arterial spurting are noted Large drops indicate blood loss was severe A cut carotid artery could easily produce the volumes of blood seen in the previous photo Insect Transfer of Blood It is possible that flies may gorge themselves on blood at a crime scene and subsequently regurgitate the blood This may give investigators an inaccurate view of the location of the bloodshed Investigators must be aware of such a possibility and take this into account when seeing seemingly unexplainable patterns Origin of the Blood • The site of origin of the blood can often be determined by drawing straight lines through the long axis of several individual bloodstains • The intersection or point of convergence or the lines represents the point from which the blood emanated Other Body Fluids- Semen • Another body fluid often searched for in cases of rape is semen • Semen is the fluid ejaculate that contains sperm and other secretions • Acid phosphatase is an enzyme present in large amounts in semen • MUP (4-methyl umbelliferyl phosphate) will flouresce under UV light when it comes in contact with acid phosphatase • Used to search large areas (bedsheets, carpets, etc.) Other Body Fluids - Semen • Also, soaking material in water and then examining the water may reveal the presence of sperm • Many men have had vasectomies, so sperm are not always present Summary • Blood testing (serology) can reveal many details about the owner including the presence of disease, toxins, and cell damage • The immune system is used to test blood for type and for drugs • Blood stains, even if old or cleaned can be detected and tested • Blood spatter patterns often reveal information about the whereabouts of the victim and/or perpetrator during the crime THE END