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Chapters 6 and 8: Blood and Forensic Medicine. Background Information Questions such as “Is this blood”, “Is this human blood” and “what biochemical markers.

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Presentation on theme: "Chapters 6 and 8: Blood and Forensic Medicine. Background Information Questions such as “Is this blood”, “Is this human blood” and “what biochemical markers."— Presentation transcript:

1 Chapters 6 and 8: Blood and Forensic Medicine

2 Background Information Questions such as “Is this blood”, “Is this human blood” and “what biochemical markers are found in the blood” are the central questions that surround blood evidence. Blood evidence became a standard piece of evidence uses to link a person with evidence left at the crime scene in the early 1900’s. Investigators were searching for something other than simply fingerprints, because fingerprints can have a lot of error due to different interpretation.

3 Background Information The uprising to fame of blood evidence can be tied to a discovery by Karl Landsteiner. He is responsible for discovering the ABO blood typing system that we still use today. Everyone has certain protein markers found on their red blood cells, that creates a signature combination… A,B, and O and “Positive” and “Negative.” We will talk about how these are determined later.

4 Background Information Land stein's discovery came long after blood transfusions were beginning to happen. Blood transfusions have been occurring for hundreds of years, however people could not understand why sometimes they failed. Blood transfusions were actually banned in Europe in the 1700’s because of their terrible success rate. Leonard Landois observed that when blood samples from two different people were mixed, they appeared to clump and form an immense amount of blood clots – which would lead to the patient dying.

5 Background Information After Landsteiner discovered ABO typing, he was able to observe which combinations of blood samples would result in compatible mixtures, and which would ultimately lead to blood clotting/clumping. It is the ABO typing that is often also used in the forensic analysis of blood samples as well.

6 Background Information In the late 1800’s, chemical tests were developed to answer the question “is it blood.” Guaiacum test: plant extract turned blue in the presence of blood. A modified version is still used today to screen for colon cancer. However, this test gave a lot of false positive and wasn’t able to distinguish between animal and human blood.

7 Cellular Components and Function of Blood Kastler – Meyer Test: Still in use today although it cannot be used to tell the difference animal and human. Also gives false positives. Phenolphthalein is used as an indicator. When mixed with hydrogen peroxide and blood, the indicator changes from colorless to pink.

8 Background Information The chemistry behind the Kastler-Meyer Test: Hydrogen Peroxide is toxic to our tissue and our hemoglobin are capable of breaking down peroxides into water and a free oxygen radical. The oxygen radical is incredibly reactive and will oxidize the indicator. The indicator was prepared by boiling the solution and adding a little zinc dust to eliminate nearly all of the oxygen in the indicator. When the oxygen radical is released from the peroxide, and when the indicator is exposed to the radical – it induces the color change from colorless to pink.

9 Background Information The Uhlenhuth test: first to distinguish human blood from animal blood. It was considered a “precipitin” test. Developed in 1900. He transfused hen’s blood into rabbits. Then when he mixed serum from the rabbit with egg white, there was a precipitate in the mixture – the egg proteins. Blood must then contain proteins that are specific to the organism and will precipitate out.

10 Cellular Components and Function of Blood Serology: body fluids – blood, saliva, urine, semen etc. Most of the time spent working with blood serum. Focused on antibody-antigen reactions and identifying which antibodies are found in the serum. Immunology: Many branches, but forensically we are concerned with detecting substances in the blood. Tests developed for blood types, microbes, drugs, toxins and disease give forensic investigators a tremendous amount of information.

11 Cellular Components and Function of Blood Fluid Dynamics: How blood flows, density, velocity and viscosity. Blood Pattern Analysis: chemistry, physics, fluid dynamics to determine what weapon, how quickly it was used, where the attack happened, where the victim and attacker were positioned.

12 Cellular Components and Function of Blood Blood is a tissue! Many components that make up it’s volume. Plasma: Liquid portion and makes up 55% of volume. Still contains fibrinogen and platelets that are responsible for clotting. Serum: After blood clots, and if the solids/clotting factors are centrifuged and removed – you are left with serum – slightly yellow in color.

13 Cellular Components and Function of Blood The plasma and blood is responsible for transporting nutrients, messenger compounds, hormones and regulate temperature. Slightly basic pH, works closely with immune system – carries antibodies, immunoglobulin's and clotting factors to sites where we need to fight off infection or repair damaged tissue.

14 Cellular Components and Function of Blood When the blood supply is cut off or diminished, we don’t have enough oxygen necessary for respiration and we build up the waste products of respiration – carbonic acid, carbon dioxide and lactic acid. Ischemia: blood supply cut off to tissue or organ – leads to significant damage and cell death.

15 Cellular Components and Function of Blood The viscosity of our blood is 3x the viscosity of water, although the same density. More resistant to flow – this will be relevant during blood pattern analysis.

16 Cellular Components and Function of Blood Erythrocytes: Red Blood cells Leucocytes: White blood cells Thrombocytes: platelets

17 Cellular Components and Function of Blood

18 Red Blood Cells: Carry oxygen from lungs to cells and return carbon dioxide from cells to lung to be eliminated from the body. Their shape – flexible, concave, disc shaped cells contributes to their function. It allows for efficient gas exchange. They contain hemoglobin which is the protein that actually carries that oxygen and gives blood it’s red color. Oxygenated blood is bright red, deoxygenated blood with too much carbon dioxide is a dark red. Blood is not blue.

19 Cellular Components and Function of Blood The red blood cell portion of our blood is sometimes referred to as hematocrit. The amount of red blood cells differs from men to women, men have a higher percentage. Red blood cells are produced in the marrow from stem cells – and as they mature they get rid of all of their organelles – making them useless for DNA analysis. They have a lifespan of 120 days and are removed by the spleen or liver.

20 Cellular Components and Functions of Blood The primary responsibility of RBC: transport oxygen and carbon dioxide using hemoglobin molecules. Carbon monoxide poisoning works by altering the hemoglobin molecules themselves. Carbon monoxide can also readily bind to hemoglobin – and form carboxyhemogloblin which is much more stable than oxyhemoglobin. The carbon monoxide is released much more slowly than the oxygen/carbon monoxide. It can be reversed if caught in time, but if not death due to asphyxiation occurs.

21 Cellular Components and Function of Blood White blood cells – general term for a large group of many different types of cells. Primary job is fighting disease, infection. Also produced in the marrow and become specific types of cells such as neutrophils, B- cells, T-cells, monocytes and natural killer cells.

22 Cellular Components and Function of Blood Platelets: aka thrombocytes: blood clotting and repair. Much smaller than RBC’s but also small discs with concave center and no nucleus. Begin in the bone marrow and are removed by the spleen after 8-10 days. They are activated when injury to a blood vessel is detected and begin a complex chemical reaction that results in sticky fibrin strands and blood cloths to form.

23 Blood Testing To answer the question: “Is it blood” many different tests have been developed – but may give false positives. In modern crime investigations, we often find it necessary to locate blood that cannot be seen and identify it as blood rather than another biological fluid. Luminol and Fluorescein: glow in the presence of trace amounts of blood.

24 Blood Testing Luminol: It glows (chemiluminescence) due to a chain of chemical reactions between the iron in the hemoglobin groups. Distinctive blue glow that is very sensitive. Some problems are that luminol will also luminesce in the presence of copper, horseradish, bleach, fecal matter, animal blood. May be a carcinogen. Requires complete darkness and is short lived – making it difficult to view and photograph.

25 Blood Testing One of the main problems with luminol testing is that the products are still soluble, so rather than being a precipitate which would stay, the solution continues to run when used on vertical surfaces – which highly distorts the bloodstain. Also – when luminol is used on horizontal non-porous surfaces – the solution of luminol and the blood will start to form pools.

26 Blood Testing Fluorescein: applied the area and then exposed to UV light. The iron of the hemoglobin causes the fluorescein to be oxidized and then fluoresce when exposed to UV light.

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29 Blood Testing The very “wet” spraying of luminol and fluorescein can distort patterns in the blood, footprints, spatter etc. Many times, a solution of the dye Leucocrystal violet is used instead: it produces a dark purple color on contact with blood. All of these tests are to see whether or not blood is anywhere in the crime scene and are considered presumptive. In order to confirm – tests called crystal tests have been developed. Takayama Test: Pyridine and glucose solution is applied to the sample – in the presence of blood, red large crystals will form. Today, we want to answer two questions at once: is it blood and whose blood is it.

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31 Blood Testing To determine whether blood is human or not, we rely on our bodies response to foreign substances and the immune response that follows. Our bodies develop antibodies to foreign substances called antigens. Antigen: any substance that can generate the production of antibodies. Antibody: A “Y” shaped protein molecule that binds with a foreign antigen to disable or destroy the substance.

32 Blood Testing Our blood contains a large amount of proteins that are specific only to human blood. When proteins or other molecules are introduced into our body that are different (antigens), this elicits an immune response and antibodies bind to the foreign proteins and often form a precipitate. These reactions between antigen and antibodies are called precipitin reactions.

33 Blood Testing These precipitin reactions are considered immunoassays due to using antibodies to identify biological samples. In order to use these to identify human blood vs. non-human blood, technicians employ anti human serum. Anti human serum will attack human blood and generate a precipitate only if the blood is human, not any other organism.

34 Blood Testing We can generate anti-serum for any protein antigen, which means we can easily identify species specific blood samples. Specifically, the ring precipitin test layers the anti- human serum in the bottom of a test tube and the extract from the blood stain on top. The anti-human antibodies will migrate towards the blood and if it is human – a precipitate ring will form at the interface.

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36 Blood Testing We can employ similar immunoassay techniques can be used to test for the presence of drugs or other substances: EMIT and ELISA EMIT test is usually used as the workplace drug testing due to it’s simplicity but it can also be used to determine what type of drug and how much was in a possible suspects system.

37 Blood Testing The drug we are testing for is attached to a protein and then injected into an animal – who will produce antibodies against this antigen. The antibodies can be isolated from the serum and now you have a substance that will react with any drug molecules in a given urine sample. Determining that the drug is present is the easy part, figuring out how much is in the system is slightly more difficult.

38 Blood Testing In order to determine how much of a substance was present, the easiest way is to determine how much of the anti-drug antibodies were used up. After the initial reaction, a complex of a drug/enzyme will be added, which will also cause a reaction with the antibodies, which will use up the remainder of the anti-drug antibodies. If we know exactly how many drug/enzyme complex we added, and we can easily determine how many are left over, we know how much of the anti-drug serum was used up fighting the second drug complex. We can then work backwards to determine how much of the anti-drug serum was used to fight off the drug that were present initially.

39 Blood Testing Another option for the second step is rather than using enzyme activity to determine how much is left over, sometimes they will tag the drug with a radioactive tracer. This is called a radioimmunoassay.

40 Blood Testing ELISA: similar method as the EMIT test however, the antibodies that are produces are attached to a surface, such as plastic in a reaction chamber. The solution containing the drug or other antigen is then passed over the surface. It will bind to the antibody and then also be attached to the reaction chamber. You can wash away everything except the antigen/antibody combo that is stuck to the wall.

41 Blood Testing ELISA continued… Another solution will then be passed over the wall of the chamber: this solution will react with any antigen-antibody compound and it uses an enzyme as a flag for where the drug is present. After everything is washed away a second time, wherever the enzyme flag is found is where the drug must have reacted with the antibody that was stuck to the wall. The substrate of the enzyme is then added, which will cause a color change and can be measured with a spectrophotometer. The more intense the color, the more drug (antigen) must have been present.

42 Blood Testing http://ccrhawaii.org/index.php/protein-techniques/32- protein-detection-a-analysis/32d-elisa/32d-animation http://www.sumanasinc.com/webcontent/animations/co ntent/ELISA.html

43 Blood Testing There are some risks of false positives, but the tests are relatively easy to administer and the reagents used are stable for a long period of time. EMIT: drugs, drug metabolites and hormones. Faster results but less sensitive. ELISA: larger proteins, such as those associated with disease. Used for HIV testing.

44 Blood Testing Monoclonal Antibodies: Our bodies respond to foreign invaders with polyclonal antibodies – meaning antibodies that each target a different part of the antigen to destroy it. However, because these tests rely on such a high level of specificity of the antibody/antigen reaction, we have been able to produce monoclonal antibodies that are uniform in nature and much more targeted than our natural immune reaction.

45 Blood Testing Finally, investigators want to know whose blood is it. Remember Karl Landsteiner: he discovered ABO typing as way to ensure that blood transfusion were more successful. He observed that when Type A blood was mixed with Type B blood, clumping occurred. But Type A blood could be mixed with other Type A and there was no problem.

46 Blood Testing Your blood type is an inherited trait. Within the genome, we all have a gene that results in the formation of glycoprotein which will stick off the surface of a red blood cell. There are three possible forms of this glycoprotein, there are three different possible appearances of the end of the protein that gives us the ABO typing.

47 Blood Testing If the glycoprotein only has the fructose exposed, this is called the H antigen and will result in an O blood type. If we take the H antigen and modify it, by adding a acetylgalactose amine: we get the A antigen = A blood type. If we take the H antigen and modify it with a galactose: B antigen, B blood type.

48 Blood Testing We inherit one copy of the gene from each parents so there are 6 genotypes which give rise to 4 possible phenotypes: Genotypes: HH, HA, AA, HB, BB, AB. Phenotypes: A and B are co dominant so the blood types are O, A, B, and AB.

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50 Blood Testing Although we may have type A blood, we are still carrying around the anti-B antibodies because some bacteria or other substances we are exposed to at a young age contains the B antigen. If type B blood is put into a Type A person, the anti-B antibodies will create an immune response and you will see the clumping or coagulation that Landsteiner observed.

51 Blood Testing This works the same in the other direction for someone with Type B blood. Type AB blood contains neither anti-A nor anti-B antibodies but has both A and B antigens on the surface: so they can accept both types of blood without an immune reaction and are considered the universal acceptor. Type O blood contains both antibodies against A and B but has neither A or B antigens on the surface. They can give to anyone but can only accept blood from other Type O people.

52 Blood Testing Determining the blood type of someone requires mixing their blood with both anti A and anti B sera and observing whether coagulation occurs or not. If A or AB is present then it will clump with anti A If B or AB is present, clump with anti B If no reaction: blood type is O.

53 Blood Typing We have many receptors and other surface molecules that can be used to distinguish between two different individuals blood type. There are over 600 blood antigens and 29 blood groups due to the different combinations of these antigens. Many of these antigens are considered Rhesus antigens – as that is the other major type of classifying blood.

54 Blood Typing When blood is described as being positive or negative, it is in reference to the presence of the D antigen on the surface. For blood transfusions, we ensure the compatibility for both the ABO and Rh factor.

55 Blood Typing Blood typing can be useful in paternity tests, although most of the time it is done through DNA testing now. Thank you Maury Povich. For example, two O parents cannot produce an A or B child. Or two A parents cannot produce a B or AB child.

56 Blood Pattern Analysis The location and the patterns of blood at the crime scene is largely influenced by two properties: Surface Tension: dependent upon intermolecular forces, and influences how droplets on surface and air are formed. Viscosity: resistance to flow. Low viscosity means it flows very easily, like water. Syrup has a high viscosity.

57 Blood Pattern Analysis Passive Bloodstains: residue of droplets, pools and flows that are formed by the action of gravity. Can either be blood dripping off a surface or blood leaving a wound and falling to the ground.

58 Blood Pattern Analysis Passive drops: droplets behaving under gravity Drip patterns: blood droplets fall into other blood Flow patterns: the shape of blood stain due to the movement of blood on a surface Pool patterns: blood that does not move on a surface for a long time.

59 Blood Pattern Analysis The shape of blood droplets can often give an indication of the angle from which it fell. Blood droplets are perfectly spherical due to their surface tension. When the hit a surface from directly above, they will form a perfectly round spot. However, when the angle changes, this changes the shape of the blood stain dramatically. Satellite droplets – small splashes – can also give you an indication of how quickly the blood was travelling when it hit the surface.

60 Blood Pattern Analysis The degree of blood drying can give investigators a clue has to how much time has passed from when the blood was left on a surface. Blood drying is influenced by the surface, how much blood is present and the environmental conditions… but most blood stains dry in a predictable fashion. They tend to dry from the perimeter inward and in the center – you will often find evidence of clotting.

61 Blood Pattern Analysis Even immediately after the blood hits the surface, it begins to dry and forms a ring around the edge and if it is left alone – the ring will begin to have a crusty texture. When wiped up, the ring is more difficult to remove than just picking up the liquid droplet, and is often left behind. Finally, the surface also influence how the droplets form: Smooth surfaces usually result in clear, sharp droplets. Rough surfaces like wood, carpet, concrete – the blood droplet will distorted.

62 Blood Pattern Analysis Active Bloodstains: bloodstains that are a result of force and gravity. Blood may be scattered off the weapon or from the blood pressure of the body pushing the blood from the site of the wound. When blood is given off from the weapon, the velocity of the droplets can be determined and classified according to their speed.

63 Blood Pattern Analysis Low Velocity: 5ft/sec. This allows the blood droplets to remain mostly spherical and typically 3 to 4 mm in diameter. Medium Velocity: 5 – 25 ft/sec. The size is usually smaller, 1-4 mm in diameter. Typically, blunt and sharp force injuries. High Velocity: 100 ft/sec, much smaller blood droplets, almost “mistlike”. Typically seen from bullet wounds, sneezing blood, coughing, or injuries from power tools.

64 Blood Pattern Analysis In addition to blood droplets being flung from weapons, arterial wounds will also be characterized as active blood stains. Arterial gush and spurts are usually easily identified by a inverted V shaped due the pumping of heart. The blood pressure is approximately 2 psi, and when arteries are severed – blood can actually be shot several feet.

65 Blood Pattern Analysis Blood coated objects will also cause blood to fly off the weapon as it being used. The resulting blood stains are referred to cast off stains or cessation. Cast off stains: created when the object is moving quickly. Cessation: caused when the object stops moving.

66 Blood Pattern Analysis By studying the droplets themselves, we are able to determine where the droplets came from, indicating where the weapon was being used or where the person may have been standing.

67 Blood Pattern Analysis When blood hits a surface at 90 o, you will see a round droplet. However, when the angle changes, the blood droplet assumes a different shape. Bloodstains that fall from an angle of less than 90 o begin to become more and more elongated. The relationship between the width and length of the droplet can give us a reasonable estimate of the angle from which it fell.

68 Blood Pattern Analysis The width of the bloodstain and the length of the bloodstain are used to construct a right triangle. We can then use trigonometry to determine the angle.

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70 The shape of the bloodstain can also give an indication of the where the bloodstain came from. The narrow end of the elongated primary droplet after hitting a surface, will point in the direction of travel. If the primary droplet hits with enough velocity to produce satellite droplets, the narrow end will point toward where the original droplet hit the surface.

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72 Both of these pieces of information can be used to determine the point of convergence, in other words, the bloodstains point of origin. Lines are drawn from each droplet and this is done for several stains. Where the lines intersect will provide a reasonable estimate as to where the droplet originated.

73 Blood Pattern Analysis Transfer Bloodstains: objects that have wet blood on them touch a surface and transfer the blood and a print onto the surface. Can be used to determine the identity, movement, identifying weapons, direction of the people involved in the incident

74 Blood Pattern Analysis At a crime scene, all blood patterns and evidence is photographed, measured and recorded. If wet blood is found on clothing or other surfaces, those items are air dried and stored in a dry paper bag. If incredibly wet, they will be stored in plastic as they are transported back to the lab, and then allowed to dry. A 10% bleach solution can be used to decontaminate blood – HIV can survive for 15 days outside the body. The blood is then refrigerated to prevent decomposition of blood components.

75 Other Biological Fluids Saliva: can be used to suggest sexual contact, recovered from used coffee cups/cigarettes and bite marks and processed for DNA testing. Presumptive tests exist to determine whether or not saliva is present in a sample, and typically rely on testing for enzymes like amylase or the epithelial cells that are constantly being shed.

76 Other Biological Fluids Presumptive Test for Saliva: Amylase is present in human saliva and is used to break down carbohydrates. The suspects saliva is mixed with starch solution, and if amylase is present, the enzyme will begin to break down the starch into simpler monosaccharide's like glucose. The mixture is then treated with iodine, which turns blue black in the presence of starch.

77 Other Biological Fluids The test can give false positives as the iodine will react with proteins that can be found in blood, semen and other fluids. Commonly, a test that uses starch attached to a dye molecule, so the reliance on iodine’s color change isn’t necessary. As amylase breaks down the starch, the dye is released and a color change is visualized.

78 Other Biological Fluids Semen: Presumptive tests is looking for high levels of Seminal Acid Phosphatase (SAP). This enzyme is produced from puberty till about 40 years old, when the production begins to decline. Brentamine Fast Blue B – turns purple in the presence of semen.

79 Other Biological Fluids Semen: confirmatory tests are typically done under a microscope and employ the use of dyes such as Nuclear Fast Red and picocarmine that are sperm cell specific. PCR techniques have been developed to detect the DNA in male sperm is very sensitive. Tests are also done using the same type of antibody reaction that is done to test for blood types.

80 Other Biological Fluids Urine: Composed of water, salts. Formed in kidneys, stored in bladder and excreted through urethra. Forensically significant when testing for the presence of compounds such as drugs, poison, alcohol. Not often tested because most tests lack sensitivity, and urine is easily absorbed into materials.


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