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but CPU-Net MEDICAL ALERT A Bi-Monthly Bulletin Published by the Child Protection Unit Network Jan – Feb 2005 Vol. 3 Issue 1 BLOOD TYPING: In Or Out As Evidence? A single blood-trace can provide a wealth of information in crime investigations. A bloodstain in a crime scene always links the suspect and victim to one another and the scene of violence. Blood and tissue forced under the fingernails of the victim during a violent struggle can also be linked to the assailant. There are specific steps in analyzing blood stains and analytical techniques have improved that would help narrow down suspects. For example, traces of drugs found in a bloodstain indicate medical treatment which a person might be receiving. Nevertheless, forensic serology is important not only for narrowing suspicion on the guilty but also in showing a suspect's innocence. Blood typing is a mere part of forensic serology but has been traditionally used in excluding suspects in certain cases. However, as this CPU-Net MEDICAL ALERT would show, blood typing has its limitations and may not be reliable even as a tool for excluding suspects. When suspicious stains are found in a crime scene or on a victim, the following are questions and tasks done by a forensic serologist: 1. Is the sample blood? The first task in examining suspicious stains is to determine whether they are blood. A number of substances such as fruitstains or dye may soil clothing and take on the appearance of bloodstains. The benzidine test is used for many years but has been discontinued because the reagent is carcinogenic. At present, the Kastle-Meyer test is used to confirm the presence of blood. The test uses phenolphthalein solution, which turns pink in contact with even small traces of blood. The test works by detecting the presence of the enzyme peroxidase in the blood. However, this substance is also present in other biological materials, such as potato and horseradish. Thus, the Kastle-Meyer test is regarded as a screening procedure. It is a highly sensitive test and a positive reaction is judged presumptive of blood. Further confirmatory tests are then carried out. These are usually chemical and microscopic procedures to identify blood by its pigments and cellular structures. 2. What if there are no detectable bloodstains? To detect invisible bloodstains, the luminol test is used, which is a chemical sprayed on carpets and furniture. This would reveal a slight phosphorescent light in the dark where bloodstains (and certain other stains) are present. 3. Is the sample animal blood? If it is animal blood, from what species? Once a stain has been confirmed as blood, it has to be determined whether it is human or animal using the precipitin test. Blood of every animal species contains different proteins, and blood from one species will develop antibodies from another species as a protective measure against disease and foreign matter. This principle is used to test whether bloodstains are human or not. Serum for the precipitin test is obtained from rabbits which have produced antibodies to destroy a small quantity of human blood injected into them. A drop of this anti-human serum is added to suspect blood, which will precipitate its protein if it is of human origin. Police laboratories hold anti-sera for most common animals, thus allowing the crime investigator to confirm or disprove statements made by the suspects about the origin of suspicious bloodstains. The precipitin test is sensitive, and will work on small traces of blood. The test is also known as the Uhlenbuth test after the German scientist who developed it in 1901. You may ask, “Why is it important to test for animal blood?” The answer is that any possibility of an injury to the household pet must be ruled out (or a fight between two pets, if pets are present). Pets normally spread human bloodstains all around the crime scene, but the pet can be a victim, perpetrator, or witness (by the transfer of animal DNA to the perpetrator). 4. If it is human blood, what is the blood group or type? To determine the blood type, forensic scientists must first determine if they have an adequate and quality sample. If so, direct typing using the A-B-O system is done. The blood type can be classified as A, B, AB or O based on the specific agglutinogen present in the red blood cells. Blood begins to dry after 3-5 minutes of exposure to air. As it dries, it changes color towards brown and black. If the blood stain has dried, indirect typing would have to be done. The most common technique used is the absorption-elution test. It is done by adding compatible antiserum antibodies to a sample, then heating the sample to break the antibody-antigen bonds, then adding known red cells from standard blood groups to see what coagulates. 5. Can the sex, age, and race of the source of blood be determined? The sex of the blood source can be determined by observing a distinctive drumstick-like structure called Barr body in white blood cells. Their appearance in blood of unknown origin is a basis for identifying it as from a female. The color of dried blood changes in time from red to brown, and the peroxidase test takes longer to develop with an old stain. It is now possible to measure color-change scientifically. Spectrophotometric analysis of bloodstains allows them to be aged within the range of one day to three weeks. Certain albeit controversial racial genetic markers involving protein and enzyme tests help determine race. The ABO system of typing blood including the Rh factor has certain proportions within a population but this is not entirely helpful. 6. Are there other tests that can be done on blood found in a crime scene? Wet blood has more value than dried blood because more tests can be run. For example, alcohol and drug content can be determined from wet blood only. DNA can be extracted from blood if white blood cells which always contain a nucleus are present. The DNA extracted and CPU-Net Phone: (632) 404-3954 Fax: (632) 404-3955 Mobile Hotline: 0917-8900445 E-mail: [email protected] URL: www.childprotection.org.ph CPU-Net MEDICAL ALERT | Jan - Feb 2005 | Volume 3, Issue 1 | Page 2 analyzed can then be matched with that of a suspect. This is the most reliable test at present that could be used in identifying the source of blood in a crime scene. 7. Can parentage be determined by blood types? x The rapid development of new genetic markers during the past 40 years has greatly enhanced our ability to identify particular people by testing their blood. As more polymorphic markers are recognized and utilized, it will undoubtedly become clear that no 2 related individuals are alike. Although blood typing was once used as a tool in determining parentage, this system is completely inconclusive. Even the most uncommon blood type is shared with at least 10% of the population, meaning millions of other men are just as likely to be the father. Elimination by blood type is also inconclusive because it does not take natural mutation into account. Application of DNA technology has now replaced blood groups and serum markers in forensic studies. 8. Are there instances when the ABO blood grouping can be compromised? Phone: (632) 404-3954 Fax: (632) 404-3955 Mobile Hotline: 0917-8900445 E-mail: [email protected] URL: www.childprotection.org.ph KLIK 2 SPEAK ¾ www.childprotection.org.ph/kubokliks x x x Unfortunately, there are situations which can lead to discrepancies in ABO blood typing. These are: Clerical error: There should be a rigorous protocol in specimen collection. This is not only important in cases of blood transfusion but also in collecting forensic evidence. We can emphasize enough that the chain of evidence not be compromised at all from collection, transport of the evidence to testing and submission of the results. Technical factors x INFECTED RBCS. Infected red cells can be agglutinated by normal human serum. These cells will test as AB when the individual is actually group O, A or B. Thus, red cells should be tested as soon as possible and stored properly as clotted specimen at 4 C. x FIBRIN CLOTS. Small fibrin clots especially in long standing specimens may appear as agglutination and read as another blood group when tested. x MISCELLANEOUS ANTIBODIES. Antibodies to the testing reagents such as dyes or preservatives may be present in the blood sample that would cause false positive agglutination reaction when testing for blood types. x LEVELS OF NATURALLY OCCURRING ANTIBODY. If a person’s antibody production is too low as in the case of newborn babies and the elderly, they may not produce enough anti-A or anti-B levels that would result in accurate blood group testing. x If blood stains are left for a long time, testing it will result in blood group “O” even if this is not the original blood type. Moreover, if there are two or more different blood types admixed in a stain, e.g. A and O, only blood type A will be identified and not the other blood types present. CHILD PROTECTION UNIT NETWORK x Discrepancies due to disease processes TRANSFUSION. If an individual has been transfused red cells from a different but compatible blood group e.g. O Rh negative cells, mixed field reactions may result. BONE MARROW TRANSPLANT. In certain cancers, a person may get a bone marrow transplant. This involves killing the person’s own bone marrow with chemotherapy then transfusing healthy bone marrow. If the blood group transfused differs from the recipient, a mixed field reaction may be observed due to mixed reaction of the person’s own residual red cells and that of the donor marrow. LEUKEMIA AND MALIGNANCIES. The levels of A and B antigens are decreased in leukemia, Hodgkin’s disease, and lymphoma. There may be mixed field reaction with ABO typing due to weakened agglutination. ACQUIRED B PHENOTYPE. Scientists have realized that the red cells can actually acquire an antigen of B type. This is caused by certain bacteria such as Proteus and Clostridium, which produce substances similar to A, B, and other blood type substances, and thus may result in false grouping. PARAPROTEINS. Myeloma and other diseases produce large amounts of abnormal proteins that can cause false agglutination reaction resulting in false blood typing. 9. Can blood not be typed? The case of the Bombay phenotype. Rare individuals with Bombay phenotype will test as Type O blood. When this blood group was first encountered, it was found not to be of either group A or B and so was thought to be of Group O. But on further test, it did not match even for O Rh negative or O Rh positive because of the absence of Antigen 'h'. Individuals with Bombay phenotype blood groups can only be transfused with blood from other Bombay phenotype individuals. Given that this condition is very rare to begin with, any person with this blood group, who needs an urgent blood transfusion, would have a huge problem as it would be quite unlikely that any blood bank would have any in stock. References: http://faculty.ncwc.edu/toconnor/425/425lect13.htm http://www.policensw.com/info/forensic/forensic6b.html http://en.wikipedia.org/wiki/Blood_type http://www.bh.rmit.edu.au/mls/subjects/abo/resources/testing5.htm Nathan D & Oski F, Hematology of infancy & childhood 4th ed, Saunders 1993 ANNOUNCEMENT CPU-Net Workshop “CHILD ABUSE (SHAKEN BABY SYNDROME) INVESTIGATION” Lecturer/Facilitator: Mr. Craig Smith, Canadian Police 4 April 2005, 8am to 5pm Traders Hotel Manila RSVP: Not later than March 21 Experts on DNA & child rights issues available on-line to answer your questions.