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FSB04 blo od spatte r All about blood Teacher Background Information Composition of blood Blood is a fluid that makes up approximately eight percent of the total weight of humans. This equates to between four and five litres in females and between five and six litres in males. (good value to know is 70ml ± 10ml per Kg body weight) The primary function of blood is to supply nutrients and elements to tissues and to remove waste products (such as carbon dioxide and lactic acid). Blood also enables cells and different substances (amino acids, fats and hormones) to be transported between tissues and organs. The normal pH of human blood is approximately 7.40 (normal range is 7.35-7.45). Blood that has a pH below 7.35 is acidic, while blood pH above 7.45 is alkaline (basic). Blood is broken into two components: the formed elements (hematocrit) and the plasma. Formed elements of the blood constitute about 40% of whole blood. The main types of formed elements in blood are red blood cells, white blood cells and platelets. (Varies considerably 25% anemia sufferers, 65% Nepalese Shirpa’s, 44-50% women, 48 -54% males – hematocrit also varies depending on the size of the vessel within the body i.e. can be as low as 15% in the capillaries). Red blood cells Red blood cells (erythrocytes) make up most (98.5%) of the cells in the blood. In every milliliter of blood, there are between five and six million red blood cells. The function of red blood cells is to transport the respiratory gases. They are a disk shape, which provides a large surface area for gas exchange and they are also flexible, which allows them to squeeze through narrow blood vessels. Red blood cells are generated by stem cells, which are found in bone marrow. Red blood cells start to break down after about 120 days of circulating around the body. Cells are cleaned up by macrophages (a type of white blood cell) when they eventually break down or rupture. White blood cells There are two broad categories of white blood cells: phagocytes and lymphocytes. Phagocytes are cells that digest waste materials. A macrophage is an important type of phagocyte. The two main types of lymphocytes are B cells and T cells. B cells are responsible for making special proteins called antibodies. T cells are involved in the immune defence against foreign or infected cells. For example: attacking a cell infected with a virus. Platelets The function of platelets is to seal leaks in blood vessels and initiate blood clotting. Platelets are only small fragments of cells but they are packed with essential enzymes and chemicals. . An injury to the lining of a blood vessel exposes collagen fibers. 2. When a platelet comes into contact with collagen fibers, the platelet is activated and becomes larger and sticky. 3. The platelet releases chemicals that activate other platelets, which starts the clotting process. Plasma The other 60% is blood plasma, a fluid that is the blood’s liquid medium, appearing yellow in colour. Plasma contains gases, ions, nutrient molecules and proteins. The proteins in the plasma have many functions. For example: albumin is the most abundant protein in human plasma. It is responsible for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. IS IT BLOOD – how to tell. Red stains are often found at the scene of a crime but are they blood? A test known as the Kastle-Meyer Color Test is a presumptive test that is used to determine if a suspect stain is blood. A presumptive test cannot confirm that the stain is blood but the test can confirm that it is not blood. FSB04 blo od spatte r All about blood Are you confused? When the colourless K-M solution is added to the red stain, it will turn a deep pink colour if blood is present. The problem is that the K-M solution also turns deep pink if it comes into contact with potatoes! What happens is, if the result is positive (deep pink colour) then further tests are performed to make sure it is really blood. How does it work? Kastle-Meyer solution contains the chemicals phenolphthalein and hydrogen peroxide. Red blood cells contain the protein haemoglobin. Haemoglobin acts as a catalyst in the reaction. Haemoglobin causes hydrogen peroxide to break down into water and oxygen. The oxygen then oxidizes or causes phenolphthalein to loose 2 hydrogen atoms turning it into an ion. The phenolphthalein ion is a pink colour. This reaction will not proceed without haemoglobin (or some substance in potatoes!) acting as the catalyst. There is another tests that can be used at the crime scene rather than sending a sample to the lab (which could take quite a lot of time). FSB04 blo od spatte r All about blood Whose blood is it? Human or animal? Once the investigator knows that the red stain is blood, the next step is to find out the source of the blood – is it from an animal or from a human? The classical test that is used to find out if blood is human or animal relies on a response from the immune system. Your immune system protects your body from bacteria and viral infections but also reacts when a foreign substance, such as a protein molecule from the blood of another animal, is introduced. This is the basis of the test known as the Precipitin Test. Blood contains protein molecules called antibodies. Antibodies are special molecules that are able to identify and then target specific foreign molecules. The foreign molecules that are targeted are known as antigens. When the immune system encounters a foreign molecule that is new to the body, a specific antibody is produced that will target the antigen. The antibody attaches or binds to the antigen and destroys or neutralises it. The antibody that is produced is specific to the antigen. How does it work? If blood serum from a human is introduced into an animal such as a rabbit, the rabbit will produce antibodies to the human blood proteins. These antibodies are called anti-human antibodies. Blood from that rabbit would therefore contain anti-human antibodies. If blood from the rabbit was then placed on blood from a crime scene, the anti-human antibodies in the rabbit’s blood would target and bind to human blood proteins if the crime scene sample is human blood. When the anti-human antibodies bind to the human blood proteins a precipitate or clot forms. The clot is evidence that the blood is of human origin. With advances in medical technology it is now possible to test what the substance is without having to resort to the laboratory. This is using a device known as the ABAcard® HemaTrace®. The HemaTrace® is a immuno-chromatographic assay for the forensic identification of human haemoglobin (Hhb). The testing procedure allows for a questioned sample to be added to the test kit following haemolysis with buffer solution. If human Hb is present within the questioned bloodstain, it will react with a mobile antihuman-antibody impregnated in the absorbent test strip (stationary phase) forming a mobile antibodyantigen complex. This mobile antibody-antigen complex then migrates (mobile phase) through the test strip to a test window where an antihuman FSB04 blo od spatte r All about blood Hb antibody is immobilised. The test works on the immune response of recognition of self or non self. To interpret results, the presence of two coloured bands, one in the test area (T) and one in the control area (C) indicates a positive result, whilst the visualisation of only one band in the control area, would indicated a negative result. The ABAcard® HemaTrace® test is a sensitive, reliable and timely “species of origin” testing process for the confirmatory identification of human blood at the crime scene. Test Strip prior to use Solution migration Positive result to Human Hb Images courtesy UWA PhD research student Mark Reynolds. FSB04 blo od spatte r All about blood Is there hidden blood? Forensic scientists (CSI) are able to use chemical tests to find out if a suspect surface contains traces of blood that may be invisible to the naked eye. The test involves the use of a chemical called luminol that produces a bright blue light when it reacts with blood. The chemical test cannot be used to relate blood back to a particular person nor can the test say with certainty that the substance is blood. This is because luminol also reacts with other substances beside blood. However, the luminol test is useful because it can locate areas where there may be tiny amounts of blood. Further tests can then be carried out on samples that have been identified by luminol, to see if it is really human blood. The luminol test When using luminol to test for blood the area of suspicion must be darkened. The luminol mixture is sprayed onto the suspect area and when the bloodstain comes into contact with the luminol there is an emission of bright blue light. The chemical reaction is an example of chemiluminescence. Chemiluminescence is a process where light is produced as a result of a chemical reaction. Got to FSB30 for the luminol movie. Images courtesy UWA PhD research student Mark Reynolds. FSB04 blo od spatte r All about blood How does it work? Luminol powder is mixed with sodium carbonate (Na2CO3) and hydrogen peroxide (H2O2) plus distilled water. This produces an alkaline or basic luminol mixture. The emission of light occurs when luminol is oxidized by an oxidant in an alkaline (basic) solution BUT this reaction will NOT occur unless a catalyst is available. A catalyst is a substance that increases the rate of a chemical reaction but is not affected or changed by the reaction. The catalyst in the luminol reaction is usually a metal. In the luminol mixture mentioned above, the sodium carbonate (Na2CO3) is added to make the luminol solution alkaline and the hydrogen peroxide (H2O2) is the oxidant (the substance that donates oxygen molecules). But remember, the reaction will not occur if the catalyst is not present. When luminol is being used to detect blood, the catalyst for the reaction is haemoglobin. Haemoglobin is a molecule that contains iron and is found in red blood cells. luminol + hydrogen peroxide oxidised luminol + LIGHT haemoglobin catalyst Chemical Structure Luminol’s (C8H7N3O2) scientific name is 5-amino-2,3-dihydro-1,4phthalazine-dione. Its molecular weight is 177.16, and it has a melting point of 319ºC-320ºC. It looks like a yellow grainy substance. Haemoglobin is the catalyst in the oxidation of luminol by hydrogen peroxide. The end product of the reaction is 3-APA is 3-aminophthalate plus light. In the reaction, an intermediary product 3-APA* is formed. 3-APA* is the excited state which means that it has higher energy than 3-APA. As the energy decreases or decays, 3-APA * becomes 3-APA and brilliant blue light is emitted.