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Forensic serology

Karl Landsteiner discovered the ABO blood group system in 1901. This was one of the most significant medical discoveries of the 20th century, as it explained why blood transfusions were sometimes fatal and allowed doctors to safely transfuse blood by matching blood types. Landsteiner recognized that not all human blood is the same, and found that blood is distinguishable by its group or type. Since then, over 100 blood factors have been identified, though the ABO system remains the most important for blood transfusions. In forensics, blood typing using antigens and antibodies was used to identify individuals until the 1990s, when DNA analysis replaced it as a more discriminating technique. Serology involves testing blood and other biological samples using the

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8-1 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-1 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Chapter 8Chapter 8 Forensic SerologyForensic Serology Courtesy of C. Fanning
8-2 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  In 1901, Karl Landsteiner announced one of theIn 1901, Karl Landsteiner announced one of the most significant discoveries of the 20most significant discoveries of the 20thth Century –Century – the typing of blood – a finding that 29 years laterthe typing of blood – a finding that 29 years later earned him a Nobel Prize.earned him a Nobel Prize.  For years, physicians had attempted to transfuseFor years, physicians had attempted to transfuse blood from one individual to another, but theirblood from one individual to another, but their efforts often ended in failure because theefforts often ended in failure because the transfused blood tended to coagulate, or clot in thetransfused blood tended to coagulate, or clot in the body of the recipient, causing instantaneous death.body of the recipient, causing instantaneous death.  Landsteiner was the first to recognize that allLandsteiner was the first to recognize that all human blood was not the same; instead, he foundhuman blood was not the same; instead, he found that blood is distinguishable by its group or type.that blood is distinguishable by its group or type. HISTORY of SerologyHISTORY of Serology
8-3 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Karl Landsteiner (1868-1943)Karl Landsteiner (1868-1943)  Out of Landsteiner’s work cameOut of Landsteiner’s work came the classification system that wethe classification system that we call the A-B-O system. Nowcall the A-B-O system. Now physicians had the key forphysicians had the key for properly matching the blood ofproperly matching the blood of a donor to that of a recipient.a donor to that of a recipient. One blood type cannot beOne blood type cannot be mixed with a different bloodmixed with a different blood type without disastroustype without disastrous consequences. This discoveryconsequences. This discovery had important implications forhad important implications for blood transfusion and theblood transfusion and the millions of lives it has sincemillions of lives it has since saved.saved.
8-4 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Landsteiner’s findings opened a new field ofLandsteiner’s findings opened a new field of research in the biological sciences. Others began toresearch in the biological sciences. Others began to pursue the identification of additional characteristicspursue the identification of additional characteristics that could further differentiate blood. By 1937, thethat could further differentiate blood. By 1937, the Rh factor in blood had been demonstrated and,Rh factor in blood had been demonstrated and, shortly thereafter, numerous blood factors or groupsshortly thereafter, numerous blood factors or groups were discovered. More than 100 different bloodwere discovered. More than 100 different blood factors have been identified. However, the ones infactors have been identified. However, the ones in the A-B-O system are still the most important forthe A-B-O system are still the most important for properly matching a donor and recipient for aproperly matching a donor and recipient for a transfusion.transfusion. Karl LandsteinerKarl Landsteiner
8-5 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Until the early 1990’s, forensic scientists focusedUntil the early 1990’s, forensic scientists focused on blood factors, such as A-B-O, as offering theon blood factors, such as A-B-O, as offering the best means for linking blood to an individual.best means for linking blood to an individual. What made these factors so attractive was that inWhat made these factors so attractive was that in theory, no two individuals, except for identicaltheory, no two individuals, except for identical twins, could be expected to have the sametwins, could be expected to have the same combination of blood factors. In other words,combination of blood factors. In other words, blood factors are controlled genetically an d haveblood factors are controlled genetically an d have the potential of being highly a highly distinctivethe potential of being highly a highly distinctive feature for personal identification.feature for personal identification.  What makes this observation so relevant is theWhat makes this observation so relevant is the great frequency of bloodstains at crime scenes,great frequency of bloodstains at crime scenes, especially crimes of the most serious nature:especially crimes of the most serious nature: homicides, assaults, and rapes.homicides, assaults, and rapes. Application to ForensicsApplication to Forensics
8-6 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  The advent of DNA technology has dramaticallyThe advent of DNA technology has dramatically altered the approach of forensic scientists towardaltered the approach of forensic scientists toward individualization of bloodstains and otherindividualization of bloodstains and other biological evidence. The search for geneticallybiological evidence. The search for genetically controlled blood factors in bloodstains has beencontrolled blood factors in bloodstains has been abandoned in favor of characterizing biologicalabandoned in favor of characterizing biological evidence by select regions of our deoxyribonucleicevidence by select regions of our deoxyribonucleic acid (DNA), which carries the body’s geneticacid (DNA), which carries the body’s genetic information. As a result, the individualization ofinformation. As a result, the individualization of dried blood and other biological evidence becomedried blood and other biological evidence become a reality and has significantly altered the role thata reality and has significantly altered the role that crime laboratories play in criminal investigation.crime laboratories play in criminal investigation. Application to ForensicsApplication to Forensics
8-7 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-7 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Nature of BloodNature of Blood •The word blood refers to a highly complex mixture of cells, enzymes, proteins, and inorganic substances. •Plasma, which is the fluid portion of blood, is composed principally of water. The plasma accounts for 55% of blood content. •Red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes) are the solid materials suspended in plasma. •Blood clots when fibrin traps and enmeshes the red blood cells. If the clotted material where removed, a pale yellowish liquid known as serum would be left. •Antigens, usually proteins, are located on the surface of red blood cells and are responsible for blood-type characteristics.
8-8 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-8 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Antigens and AntibodiesAntigens and Antibodies • Red blood cells transport oxygen from the lungs to the gody tissues and remove carbon dioxide from tissues by transporting it back to the lungs, where it is exhales. For reasons unrelated to the red blood cell’s transporting mission, on the surface of each cell are millions of characteristic chemical structures called antigens. More than 15 blood antigen systems have been identified, but the A-B-O and Rh systems are the most important. •An individual that is type A has A antigens on his/her red blood cells, type B has B antigens, AB has both A and B antigens, and type O has neither A nor B antigens. Cont.
8-9 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Another important blood antigen hasAnother important blood antigen has been designated as thebeen designated as the Rh factorRh factor,, or Dor D antigen. People with the D antigen areantigen. People with the D antigen are said to besaid to be Rh positiveRh positive; those without this; those without this antigen areantigen are Rh negativeRh negative..  In routine blood banking, the presence orIn routine blood banking, the presence or absence of the three antigens - A, B, andabsence of the three antigens - A, B, and D - must be determined in testingD - must be determined in testing compatibility of the donor and recipient.compatibility of the donor and recipient. Antigens and AntibodiesAntigens and Antibodies
8-10 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-10 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Blood TypingBlood Typing •Serum is important because it contains proteins known as antibodies. The fundamental principal of blood typing is that for every antigen, there exists a specific antibody. Each antibody symbol contains the prefix anti-, followed by the name of the antigen for which is it specific. •The serum-containing antibody is referred to as the antiserum, meaning a serum that reacts against something (antigens). •Antibodies are normally bivalent – that is, they have two reactive sites. This means that each antibody can simultaneously be attached to antigens located on two different red blood cells. This creates a vast network of cross-linked cells usually seen as clumping or agglutination.
8-11 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-11 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein SerologySerology •The term serology is used to describe a broad scope of laboratory tests that use specific antigen and serum antibody reactions. •The identity of each of the four A-B-O blood groups can be established by testing the blood with anti-A and anti-B sera.
8-12 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-12 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Serology Antigen-Antibody ReactionSerology Antigen-Antibody Reaction •The concept of specific antigen–antibody reactions has been applied to immunoassay techniques for the detection of drugs of abuse in blood and urine. Red blood cells containing A antigens do not combine with B antibodies. Red blood cells containing B antigens are agglutinated or clumped together in the presence of B antibodies.
8-13 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Let’s look a little more closely at this phenomenon. InLet’s look a little more closely at this phenomenon. In normal blood, antigens on red blood cells andnormal blood, antigens on red blood cells and antibodies coexist without destroying each otherantibodies coexist without destroying each other because the antibodies present are not specific towardbecause the antibodies present are not specific toward any of the antigens. However, suppose a foreign serumany of the antigens. However, suppose a foreign serum added to the blood introduces a new antibody. Thisadded to the blood introduces a new antibody. This results in a specific antigen-antibody reaction thatresults in a specific antigen-antibody reaction that immediately causes the red blood cells to link together,immediately causes the red blood cells to link together, or agglutinate.or agglutinate. Serology Antigen-Antibody ReactionSerology Antigen-Antibody Reaction
8-14 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Nature has taken this situation into account,Nature has taken this situation into account, because when we examine the serum of typebecause when we examine the serum of type A blood, we find anti-B and no anti-A.A blood, we find anti-B and no anti-A. Similarly, type B blood contains only anti-A,Similarly, type B blood contains only anti-A, type O blood contains both anti-A and anti-B,type O blood contains both anti-A and anti-B, and type AB blood contains neither anti-A orand type AB blood contains neither anti-A or anti-B. The antigen and antibodyanti-B. The antigen and antibody components of normal blood are summarizedcomponents of normal blood are summarized in the table given to you in your lab….in the table given to you in your lab….  Lab #1: Training Lab: Blood TypesLab #1: Training Lab: Blood Types Serology Antigen-Antibody ReactionSerology Antigen-Antibody Reaction
8-15 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Serology involves a broad scope of laboratory tests thatSerology involves a broad scope of laboratory tests that use specific antigen and serum antibody reactions.use specific antigen and serum antibody reactions.  An antibody reacts or agglutinates only with its specificAn antibody reacts or agglutinates only with its specific antigen. The concept of specific antigen-antibodyantigen. The concept of specific antigen-antibody reactions has been applied to techniques for detectingreactions has been applied to techniques for detecting abused drugs in blood and urine.abused drugs in blood and urine.  Every red blood cell contains either an A antigen, a BEvery red blood cell contains either an A antigen, a B antigen, or no antigens. The type of antigen on one’s redantigen, or no antigens. The type of antigen on one’s red blood cells determines one’s ABO blood type. Peopleblood cells determines one’s ABO blood type. People with type A blood have A antigens on their red bloodwith type A blood have A antigens on their red blood cells, those with type B blood have B antigens, and thosecells, those with type B blood have B antigens, and those with type O blood have no antigens on their red bloodwith type O blood have no antigens on their red blood cells.cells. Quick ReviewQuick Review
8-16 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Radioimmunoassay and EMITRadioimmunoassay and EMIT ImmunoassayImmunoassay TechniquesTechniques
8-17 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  The concept of a specific antigen-antibody reaction isThe concept of a specific antigen-antibody reaction is finding application in other areas unrelated to bloodfinding application in other areas unrelated to blood typing. Most significantly, this approach has beentyping. Most significantly, this approach has been extended to the detection of drugs in blood and urine.extended to the detection of drugs in blood and urine.  Antibodies that react with drugs do not existAntibodies that react with drugs do not exist naturally; however, they can be produced in animalsnaturally; however, they can be produced in animals such as rabbits by first combining the drug with asuch as rabbits by first combining the drug with a protein and injecting this combination into the animal.protein and injecting this combination into the animal. This drug-protein complex acts as an antigenThis drug-protein complex acts as an antigen stimulating the animal to produce antibodies.stimulating the animal to produce antibodies. Immunoassay TechniquesImmunoassay Techniques
8-18 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Drug Protein Carrier attached to drug Inject into rabbit Rabbit produces antibodies nearly specific to the drug. The recovered blood serum of the animal now contains antibodies that are specific or nearly specific to the drug originally attached to the protein carrier. Currently, thousands of individuals regularly submit to urinalysis for the presence of abused drugs. These individuals include military personnel, transportation industry employees, police and corrections personnel, and subjects requiring pre-employment drug screening. Immunoassay testing for drugs has proven quite suitable for handling the large volume of specimens that must be rapidly analyzed for drug content on a daily basis.
8-19 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-19 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein ImmunoassayImmunoassay •A number of immunological assay techniques are commercially available for detecting drugs through antigen-antibody reaction. •One such technique, the enzyme-multiplied immunoassay technique (EMIT), is used by toxicologists because of its speed and high sensitivity for detecting drugs in urine. •In a typical EMIT analysis, antibodies that will bind to a specific drug are added to the subject’s urine. •Other immunoassay procedures are also available, such as radioimmunoassay (RIA), which uses drugs labeled with radioactive tags.
8-20 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  EMIT stands forEMIT stands for enzyme-multipliedenzyme-multiplied immunoassay techniqueimmunoassay technique. EMIT has. EMIT has gained widespread popularity amonggained widespread popularity among toxicologists because of its speed andtoxicologists because of its speed and high sensitivity for detecting drugs inhigh sensitivity for detecting drugs in urine.urine. EMITEMIT
8-21 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-21 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Antigen-Antibody ReactionAntigen-Antibody Reaction •When an animal, such as a rabbit or mouse, is injected with an antigen its body will produce a series of different antibodies, all of which are designed to attack some particular site on the antigen of interest.
8-22 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-22 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Antigen-Antibody ReactionAntigen-Antibody Reaction •This collection of antibodies is known as polyclonal antibodies. •Alternately, a more uniform and specific collection of antibodies designed to combine with a single antigen site can be manufactured. •Such antibodies are known as monoclonals.
8-23 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Color TestsColor Tests ForensicForensic Characterization ofCharacterization of BloodstainsBloodstains
8-24 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-24 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Forensics of BloodForensics of Blood •The criminalist must be prepared to answer the following questions when examining dried blood: 1. Is it blood? 2. From what species did the blood originate? 3. If the blood is of human origin, how closely can it be associated to a particular individual? •The determination of blood is best made by means of a preliminary color test.
8-25 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-25 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Testing for BloodTesting for Blood •A positive result from the Kastle-Meyer (phenolphthalein) color test is highly indicative of blood. Hemoglobin causes a deep pink color. •Alternatively, the luminol test is used to search out trace amounts of blood located at crime scenes. •Luminol produces light (luminescence) in a darkened area. Courtesy of C. Fanning Courtesy of C. Fanning Luminol with false positive (bleach) Courtesy of C. Fanning
8-26 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  For many years, the most common test was the benzidineFor many years, the most common test was the benzidine color test, but because benzidine has been identified as acolor test, but because benzidine has been identified as a known carcinogen, its use has generally been discontinuedknown carcinogen, its use has generally been discontinued and replaced with the Kastle-Meyer test.and replaced with the Kastle-Meyer test.  Both the benzidine and the Kastle-Meyer color tests areBoth the benzidine and the Kastle-Meyer color tests are based on the observation that blood hemoglobin possessesbased on the observation that blood hemoglobin possesses peroxidase-like activity. Peroxidases are enzymes thatperoxidase-like activity. Peroxidases are enzymes that accelerate the oxidation of several classes of organicaccelerate the oxidation of several classes of organic compounds when combined with peroxides. For example,compounds when combined with peroxides. For example, when a bloodstain, phenolphthalein reagent, and hydrogenwhen a bloodstain, phenolphthalein reagent, and hydrogen peroxide are mixed together, oxidation of the hemoglobin inperoxide are mixed together, oxidation of the hemoglobin in the blood produces a deep pink color.the blood produces a deep pink color. Benzidine and Kastle-MeyerBenzidine and Kastle-Meyer
8-27 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Field investigators haveField investigators have found Hemastix strips afound Hemastix strips a useful presumptive fielduseful presumptive field test for blood. Designed astest for blood. Designed as a urine dipstick test fora urine dipstick test for blood, the strip can beblood, the strip can be moistened with distilledmoistened with distilled water and placed in contactwater and placed in contact with a suspect bloodstain.with a suspect bloodstain. The appearance of a greenThe appearance of a green color indicates blood.color indicates blood. HemastixHemastix
8-28 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Another important presumptiveAnother important presumptive identification test for blood is theidentification test for blood is the luminol test. Unlike the benzidineluminol test. Unlike the benzidine and Kastle-Meyer tests, the reactionand Kastle-Meyer tests, the reaction of luminol with blood produces lightof luminol with blood produces light rather than color. By sprayingrather than color. By spraying luminol reagent onto a suspect item,luminol reagent onto a suspect item, investigators can quickly screen largeinvestigators can quickly screen large areas for bloodstains. The sprayedareas for bloodstains. The sprayed objects must be located in aobjects must be located in a darkened area while being viewed fordarkened area while being viewed for the emission of light (luminescence);the emission of light (luminescence); any bloodstains produce a faint blueany bloodstains produce a faint blue glow.glow. Luminol and BluestarLuminol and Bluestar
8-29 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ The identification of blood can be madeThe identification of blood can be made more specific if microcrystalline testsmore specific if microcrystalline tests are performed on the material.are performed on the material. Several tests are available; the twoSeveral tests are available; the two most popular ones are the Takayamamost popular ones are the Takayama and Teichmann tests. Both depend onand Teichmann tests. Both depend on the addition of specific chemicals tothe addition of specific chemicals to the blood to form characteristicthe blood to form characteristic crystals containing hemoglobincrystals containing hemoglobin derivatives. Crystal tests are far lessderivatives. Crystal tests are far less sensitive than color tests for bloodsensitive than color tests for blood identification and are more susceptibleidentification and are more susceptible to interference from contaminants thatto interference from contaminants that may be present in the stain.may be present in the stain. Microcrystalline TestMicrocrystalline Test
8-30 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Once the stain has been characterized as blood, the serologistOnce the stain has been characterized as blood, the serologist determines whether the blood is of human or animal origin.determines whether the blood is of human or animal origin. The standard test is the precipitin test. Precipitin tests are basedThe standard test is the precipitin test. Precipitin tests are based on the fact that when animals (usually rabbits) are injected withon the fact that when animals (usually rabbits) are injected with human blood, antibodies form that react with the invadinghuman blood, antibodies form that react with the invading human blood to neutralize its presence. The investigator canhuman blood to neutralize its presence. The investigator can recover these antibodies by bleeding the animal and isolatingrecover these antibodies by bleeding the animal and isolating the blood serum, which contains antibodies that specificallythe blood serum, which contains antibodies that specifically react with human antigens. For this reason, the serum is knownreact with human antigens. For this reason, the serum is known as human antiserum. In the same manner, by injecting rabbitsas human antiserum. In the same manner, by injecting rabbits with the blood of other known animals, virtually any kind ofwith the blood of other known animals, virtually any kind of animal antiserum can be produced. Antiserums areanimal antiserum can be produced. Antiserums are commercially available for humans for human s and for acommercially available for humans for human s and for a variety of commonly encountered animals – for example, dogs,variety of commonly encountered animals – for example, dogs, cats, chickens and deer.cats, chickens and deer. Precipitin TestPrecipitin Test
8-31 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Precipitin TestPrecipitin Test  Several techniques have beenSeveral techniques have been devised for performingdevised for performing precipitin tests on bloodstains.precipitin tests on bloodstains. The classic method is to layerThe classic method is to layer an extract of the bloodstain onan extract of the bloodstain on top of the human antiserum intop of the human antiserum in a capillary tube. Human blood,a capillary tube. Human blood, or for that matter, any proteinor for that matter, any protein of human origin in the extract,of human origin in the extract, reacts specifically withreacts specifically with antibodies present in theantibodies present in the antiserum, and indicated by theantiserum, and indicated by the formation of a cloudy ring orformation of a cloudy ring or band at the interface of the twoband at the interface of the two liquids.liquids.
8-32 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Another method, calledAnother method, called gel diffusion,gel diffusion, takes advantagetakes advantage of the fact that antibodies and antigens diffuse orof the fact that antibodies and antigens diffuse or move toward one another on a plate coated with amove toward one another on a plate coated with a gel medium from a natural polymer called agar.gel medium from a natural polymer called agar. The extracted bloodstain and the human antiserumThe extracted bloodstain and the human antiserum are placed in separate holes opposite each other onare placed in separate holes opposite each other on the gel. If the blood is human, a line of precipitationthe gel. If the blood is human, a line of precipitation forms where the antigens and antibodies meet.forms where the antigens and antibodies meet. Gel DiffusionGel Diffusion
8-33 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Gel DiffusionGel Diffusion  Similarly, the antigens andSimilarly, the antigens and antibodies can be induced toantibodies can be induced to move toward one anothermove toward one another under the influence of anunder the influence of an electrical field. In theelectrical field. In the electrophoretic method, anelectrophoretic method, an electrical potential is applied toelectrical potential is applied to the gel medium; a specificthe gel medium; a specific antigen-antibody reaction isantigen-antibody reaction is denoted by a line of precitationdenoted by a line of precitation formed between the holeformed between the hole containing the blood extractcontaining the blood extract and the hole containing theand the hole containing the human antiserum.human antiserum.
8-34 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-34 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein The precipitin test is very sensitive and requires only a small amount of blood for testing. Human bloodstains dried for 10 – 15 years and longer may still give a positive precipitin reaction. Even extracts of tissue from mummies four to five thousand years old have given positive reactions with this test. Furthermore, human bloodstains diluted by washing in water and left with only a faint color may still yield a positive precipitin reaction.
8-35 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Once it has been determined that the bloodstainOnce it has been determined that the bloodstain is human, an effort must be made to associate oris human, an effort must be made to associate or disassociate the stain with a particulardisassociate the stain with a particular individual. Until the mid-1990’s, routineindividual. Until the mid-1990’s, routine characterization of bloodstains included thecharacterization of bloodstains included the determination of A-B-O types; however, thedetermination of A-B-O types; however, the widespread use of DNA profiling or typing haswidespread use of DNA profiling or typing has relegated this subject to one of historical interestrelegated this subject to one of historical interest only.only. Precipitin TestPrecipitin Test
8-36 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • The precipitin test uses antisera normally derived fromThe precipitin test uses antisera normally derived from rabbits that have been injected with the blood of a knownrabbits that have been injected with the blood of a known animal to determine the species origin of a questionedanimal to determine the species origin of a questioned bloodstain.bloodstain. • Once it has been determined that the bloodstain is ofOnce it has been determined that the bloodstain is of human origin, an effort must be made to associate orhuman origin, an effort must be made to associate or dissociate the stain with a particular individual.dissociate the stain with a particular individual. • DNA analysis has allowed forensic scientists to associateDNA analysis has allowed forensic scientists to associate blood to a single individual.blood to a single individual. Recap
8-37 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • Prior to the advent of DNA typing, bloodstains werePrior to the advent of DNA typing, bloodstains were linked to a source by A-B-O typing and thelinked to a source by A-B-O typing and the characterization of polymorphic blood enzymes andcharacterization of polymorphic blood enzymes and proteins.proteins. • This approach has now been supplanted by the newerThis approach has now been supplanted by the newer DNA technology.DNA technology. • DNA analysis has allowed forensic scientists to associateDNA analysis has allowed forensic scientists to associate blood and semen stains to a single individual.blood and semen stains to a single individual. A-B-O vs. DNA
8-38 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • The transmission of hereditary material is accomplished byThe transmission of hereditary material is accomplished by means of microscopic units called genes, located onmeans of microscopic units called genes, located on chromosomes.chromosomes. • Alternative forms of genes that influence a givenAlternative forms of genes that influence a given characteristic (such as eye color or blood type) are known ascharacteristic (such as eye color or blood type) are known as alleles.alleles. • Paternity testing has historically involved the A-B-O bloodPaternity testing has historically involved the A-B-O blood typing system, along with blood factors other than A-B-O.typing system, along with blood factors other than A-B-O. • Currently, paternity testing has implemented DNA testCurrently, paternity testing has implemented DNA test procedures that can raise the odds of establishing paternityprocedures that can raise the odds of establishing paternity beyond 99 percent.beyond 99 percent. Heredity and PaternityHeredity and Paternity
8-39 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • Many of the cases sent to a forensic laboratoryMany of the cases sent to a forensic laboratory involve sexual offenses, making it necessary toinvolve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains.examine exhibits for the presence of seminal stains. • The best way to locate and at the same timeThe best way to locate and at the same time characterize a seminal stain is to perform the acidcharacterize a seminal stain is to perform the acid phosphatase (an enzyme secreted into seminal fluid)phosphatase (an enzyme secreted into seminal fluid) color test.color test.  A purple color indicates acid phosphataseA purple color indicates acid phosphatase enzyme.enzyme. Testing for Seminal StainsTesting for Seminal Stains
8-40 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Testing for Seminal StainsTesting for Seminal Stains Semen can be unequivocally identified by either the presence of spermatozoa or of p30, a protein unique to seminal plasma. Forensic scientists can successfully link seminal material to an individual by DNA typing.
8-41 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • The rape victim must undergo a medical examination asThe rape victim must undergo a medical examination as soon as possible after the assault.soon as possible after the assault. • At that time the appropriate items of physical evidenceAt that time the appropriate items of physical evidence including clothing, hairs, and vaginal and rectal swabsincluding clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination.can be collected for subsequent laboratory examination. • All outer and undergarments should be carefullyAll outer and undergarments should be carefully removed and packaged separately in paper (not plastic)removed and packaged separately in paper (not plastic) bags.bags. • Bedding, or the object upon which the assault took place,Bedding, or the object upon which the assault took place, may also be carefully collected.may also be carefully collected. Rape EvidenceRape Evidence
8-42 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • If a suspect is apprehended within 24 hours of the assault,If a suspect is apprehended within 24 hours of the assault, it may be possible to detect the victim’s DNA on theit may be possible to detect the victim’s DNA on the male’s underwear or on a penile swab of the suspect.male’s underwear or on a penile swab of the suspect. • Items routinely collected from the suspect include allItems routinely collected from the suspect include all clothing, pubic hair, head hair, penile swab, and a bloodclothing, pubic hair, head hair, penile swab, and a blood sample or buccal swab for DNA typing.sample or buccal swab for DNA typing. • The forceful physical contact between victim and assailantThe forceful physical contact between victim and assailant may result in a transfer of such physical evidence ofmay result in a transfer of such physical evidence of blood, semen, saliva, hairs, and fibers.blood, semen, saliva, hairs, and fibers. Rape EvidenceRape Evidence

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Forensic serology

  • 1.
    8-1 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-1 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Chapter 8Chapter 8 Forensic SerologyForensic Serology Courtesy of C. Fanning
  • 2.
    8-2 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  In 1901, Karl Landsteiner announced one of theIn 1901, Karl Landsteiner announced one of the most significant discoveries of the 20most significant discoveries of the 20thth Century –Century – the typing of blood – a finding that 29 years laterthe typing of blood – a finding that 29 years later earned him a Nobel Prize.earned him a Nobel Prize.  For years, physicians had attempted to transfuseFor years, physicians had attempted to transfuse blood from one individual to another, but theirblood from one individual to another, but their efforts often ended in failure because theefforts often ended in failure because the transfused blood tended to coagulate, or clot in thetransfused blood tended to coagulate, or clot in the body of the recipient, causing instantaneous death.body of the recipient, causing instantaneous death.  Landsteiner was the first to recognize that allLandsteiner was the first to recognize that all human blood was not the same; instead, he foundhuman blood was not the same; instead, he found that blood is distinguishable by its group or type.that blood is distinguishable by its group or type. HISTORY of SerologyHISTORY of Serology
  • 3.
    8-3 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Karl Landsteiner (1868-1943)Karl Landsteiner (1868-1943)  Out of Landsteiner’s work cameOut of Landsteiner’s work came the classification system that wethe classification system that we call the A-B-O system. Nowcall the A-B-O system. Now physicians had the key forphysicians had the key for properly matching the blood ofproperly matching the blood of a donor to that of a recipient.a donor to that of a recipient. One blood type cannot beOne blood type cannot be mixed with a different bloodmixed with a different blood type without disastroustype without disastrous consequences. This discoveryconsequences. This discovery had important implications forhad important implications for blood transfusion and theblood transfusion and the millions of lives it has sincemillions of lives it has since saved.saved.
  • 4.
    8-4 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Landsteiner’s findings opened a new field ofLandsteiner’s findings opened a new field of research in the biological sciences. Others began toresearch in the biological sciences. Others began to pursue the identification of additional characteristicspursue the identification of additional characteristics that could further differentiate blood. By 1937, thethat could further differentiate blood. By 1937, the Rh factor in blood had been demonstrated and,Rh factor in blood had been demonstrated and, shortly thereafter, numerous blood factors or groupsshortly thereafter, numerous blood factors or groups were discovered. More than 100 different bloodwere discovered. More than 100 different blood factors have been identified. However, the ones infactors have been identified. However, the ones in the A-B-O system are still the most important forthe A-B-O system are still the most important for properly matching a donor and recipient for aproperly matching a donor and recipient for a transfusion.transfusion. Karl LandsteinerKarl Landsteiner
  • 5.
    8-5 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Until the early 1990’s, forensic scientists focusedUntil the early 1990’s, forensic scientists focused on blood factors, such as A-B-O, as offering theon blood factors, such as A-B-O, as offering the best means for linking blood to an individual.best means for linking blood to an individual. What made these factors so attractive was that inWhat made these factors so attractive was that in theory, no two individuals, except for identicaltheory, no two individuals, except for identical twins, could be expected to have the sametwins, could be expected to have the same combination of blood factors. In other words,combination of blood factors. In other words, blood factors are controlled genetically an d haveblood factors are controlled genetically an d have the potential of being highly a highly distinctivethe potential of being highly a highly distinctive feature for personal identification.feature for personal identification.  What makes this observation so relevant is theWhat makes this observation so relevant is the great frequency of bloodstains at crime scenes,great frequency of bloodstains at crime scenes, especially crimes of the most serious nature:especially crimes of the most serious nature: homicides, assaults, and rapes.homicides, assaults, and rapes. Application to ForensicsApplication to Forensics
  • 6.
    8-6 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  The advent of DNA technology has dramaticallyThe advent of DNA technology has dramatically altered the approach of forensic scientists towardaltered the approach of forensic scientists toward individualization of bloodstains and otherindividualization of bloodstains and other biological evidence. The search for geneticallybiological evidence. The search for genetically controlled blood factors in bloodstains has beencontrolled blood factors in bloodstains has been abandoned in favor of characterizing biologicalabandoned in favor of characterizing biological evidence by select regions of our deoxyribonucleicevidence by select regions of our deoxyribonucleic acid (DNA), which carries the body’s geneticacid (DNA), which carries the body’s genetic information. As a result, the individualization ofinformation. As a result, the individualization of dried blood and other biological evidence becomedried blood and other biological evidence become a reality and has significantly altered the role thata reality and has significantly altered the role that crime laboratories play in criminal investigation.crime laboratories play in criminal investigation. Application to ForensicsApplication to Forensics
  • 7.
    8-7 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-7 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Nature of BloodNature of Blood •The word blood refers to a highly complex mixture of cells, enzymes, proteins, and inorganic substances. •Plasma, which is the fluid portion of blood, is composed principally of water. The plasma accounts for 55% of blood content. •Red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes) are the solid materials suspended in plasma. •Blood clots when fibrin traps and enmeshes the red blood cells. If the clotted material where removed, a pale yellowish liquid known as serum would be left. •Antigens, usually proteins, are located on the surface of red blood cells and are responsible for blood-type characteristics.
  • 8.
    8-8 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-8 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Antigens and AntibodiesAntigens and Antibodies • Red blood cells transport oxygen from the lungs to the gody tissues and remove carbon dioxide from tissues by transporting it back to the lungs, where it is exhales. For reasons unrelated to the red blood cell’s transporting mission, on the surface of each cell are millions of characteristic chemical structures called antigens. More than 15 blood antigen systems have been identified, but the A-B-O and Rh systems are the most important. •An individual that is type A has A antigens on his/her red blood cells, type B has B antigens, AB has both A and B antigens, and type O has neither A nor B antigens. Cont.
  • 9.
    8-9 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Another important blood antigen hasAnother important blood antigen has been designated as thebeen designated as the Rh factorRh factor,, or Dor D antigen. People with the D antigen areantigen. People with the D antigen are said to besaid to be Rh positiveRh positive; those without this; those without this antigen areantigen are Rh negativeRh negative..  In routine blood banking, the presence orIn routine blood banking, the presence or absence of the three antigens - A, B, andabsence of the three antigens - A, B, and D - must be determined in testingD - must be determined in testing compatibility of the donor and recipient.compatibility of the donor and recipient. Antigens and AntibodiesAntigens and Antibodies
  • 10.
    8-10 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-10 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Blood TypingBlood Typing •Serum is important because it contains proteins known as antibodies. The fundamental principal of blood typing is that for every antigen, there exists a specific antibody. Each antibody symbol contains the prefix anti-, followed by the name of the antigen for which is it specific. •The serum-containing antibody is referred to as the antiserum, meaning a serum that reacts against something (antigens). •Antibodies are normally bivalent – that is, they have two reactive sites. This means that each antibody can simultaneously be attached to antigens located on two different red blood cells. This creates a vast network of cross-linked cells usually seen as clumping or agglutination.
  • 11.
    8-11 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-11 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein SerologySerology •The term serology is used to describe a broad scope of laboratory tests that use specific antigen and serum antibody reactions. •The identity of each of the four A-B-O blood groups can be established by testing the blood with anti-A and anti-B sera.
  • 12.
    8-12 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-12 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Serology Antigen-Antibody ReactionSerology Antigen-Antibody Reaction •The concept of specific antigen–antibody reactions has been applied to immunoassay techniques for the detection of drugs of abuse in blood and urine. Red blood cells containing A antigens do not combine with B antibodies. Red blood cells containing B antigens are agglutinated or clumped together in the presence of B antibodies.
  • 13.
    8-13 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Let’s look a little more closely at this phenomenon. InLet’s look a little more closely at this phenomenon. In normal blood, antigens on red blood cells andnormal blood, antigens on red blood cells and antibodies coexist without destroying each otherantibodies coexist without destroying each other because the antibodies present are not specific towardbecause the antibodies present are not specific toward any of the antigens. However, suppose a foreign serumany of the antigens. However, suppose a foreign serum added to the blood introduces a new antibody. Thisadded to the blood introduces a new antibody. This results in a specific antigen-antibody reaction thatresults in a specific antigen-antibody reaction that immediately causes the red blood cells to link together,immediately causes the red blood cells to link together, or agglutinate.or agglutinate. Serology Antigen-Antibody ReactionSerology Antigen-Antibody Reaction
  • 14.
    8-14 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Nature has taken this situation into account,Nature has taken this situation into account, because when we examine the serum of typebecause when we examine the serum of type A blood, we find anti-B and no anti-A.A blood, we find anti-B and no anti-A. Similarly, type B blood contains only anti-A,Similarly, type B blood contains only anti-A, type O blood contains both anti-A and anti-B,type O blood contains both anti-A and anti-B, and type AB blood contains neither anti-A orand type AB blood contains neither anti-A or anti-B. The antigen and antibodyanti-B. The antigen and antibody components of normal blood are summarizedcomponents of normal blood are summarized in the table given to you in your lab….in the table given to you in your lab….  Lab #1: Training Lab: Blood TypesLab #1: Training Lab: Blood Types Serology Antigen-Antibody ReactionSerology Antigen-Antibody Reaction
  • 15.
    8-15 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Serology involves a broad scope of laboratory tests thatSerology involves a broad scope of laboratory tests that use specific antigen and serum antibody reactions.use specific antigen and serum antibody reactions.  An antibody reacts or agglutinates only with its specificAn antibody reacts or agglutinates only with its specific antigen. The concept of specific antigen-antibodyantigen. The concept of specific antigen-antibody reactions has been applied to techniques for detectingreactions has been applied to techniques for detecting abused drugs in blood and urine.abused drugs in blood and urine.  Every red blood cell contains either an A antigen, a BEvery red blood cell contains either an A antigen, a B antigen, or no antigens. The type of antigen on one’s redantigen, or no antigens. The type of antigen on one’s red blood cells determines one’s ABO blood type. Peopleblood cells determines one’s ABO blood type. People with type A blood have A antigens on their red bloodwith type A blood have A antigens on their red blood cells, those with type B blood have B antigens, and thosecells, those with type B blood have B antigens, and those with type O blood have no antigens on their red bloodwith type O blood have no antigens on their red blood cells.cells. Quick ReviewQuick Review
  • 16.
    8-16 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Radioimmunoassay and EMITRadioimmunoassay and EMIT ImmunoassayImmunoassay TechniquesTechniques
  • 17.
    8-17 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  The concept of a specific antigen-antibody reaction isThe concept of a specific antigen-antibody reaction is finding application in other areas unrelated to bloodfinding application in other areas unrelated to blood typing. Most significantly, this approach has beentyping. Most significantly, this approach has been extended to the detection of drugs in blood and urine.extended to the detection of drugs in blood and urine.  Antibodies that react with drugs do not existAntibodies that react with drugs do not exist naturally; however, they can be produced in animalsnaturally; however, they can be produced in animals such as rabbits by first combining the drug with asuch as rabbits by first combining the drug with a protein and injecting this combination into the animal.protein and injecting this combination into the animal. This drug-protein complex acts as an antigenThis drug-protein complex acts as an antigen stimulating the animal to produce antibodies.stimulating the animal to produce antibodies. Immunoassay TechniquesImmunoassay Techniques
  • 18.
    8-18 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Drug Protein Carrier attached to drug Inject into rabbit Rabbit produces antibodies nearly specific to the drug. The recovered blood serum of the animal now contains antibodies that are specific or nearly specific to the drug originally attached to the protein carrier. Currently, thousands of individuals regularly submit to urinalysis for the presence of abused drugs. These individuals include military personnel, transportation industry employees, police and corrections personnel, and subjects requiring pre-employment drug screening. Immunoassay testing for drugs has proven quite suitable for handling the large volume of specimens that must be rapidly analyzed for drug content on a daily basis.
  • 19.
    8-19 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-19 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein ImmunoassayImmunoassay •A number of immunological assay techniques are commercially available for detecting drugs through antigen-antibody reaction. •One such technique, the enzyme-multiplied immunoassay technique (EMIT), is used by toxicologists because of its speed and high sensitivity for detecting drugs in urine. •In a typical EMIT analysis, antibodies that will bind to a specific drug are added to the subject’s urine. •Other immunoassay procedures are also available, such as radioimmunoassay (RIA), which uses drugs labeled with radioactive tags.
  • 20.
    8-20 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  EMIT stands forEMIT stands for enzyme-multipliedenzyme-multiplied immunoassay techniqueimmunoassay technique. EMIT has. EMIT has gained widespread popularity amonggained widespread popularity among toxicologists because of its speed andtoxicologists because of its speed and high sensitivity for detecting drugs inhigh sensitivity for detecting drugs in urine.urine. EMITEMIT
  • 21.
    8-21 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-21 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Antigen-Antibody ReactionAntigen-Antibody Reaction •When an animal, such as a rabbit or mouse, is injected with an antigen its body will produce a series of different antibodies, all of which are designed to attack some particular site on the antigen of interest.
  • 22.
    8-22 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-22 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Antigen-Antibody ReactionAntigen-Antibody Reaction •This collection of antibodies is known as polyclonal antibodies. •Alternately, a more uniform and specific collection of antibodies designed to combine with a single antigen site can be manufactured. •Such antibodies are known as monoclonals.
  • 23.
    8-23 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Color TestsColor Tests ForensicForensic Characterization ofCharacterization of BloodstainsBloodstains
  • 24.
    8-24 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-24 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Forensics of BloodForensics of Blood •The criminalist must be prepared to answer the following questions when examining dried blood: 1. Is it blood? 2. From what species did the blood originate? 3. If the blood is of human origin, how closely can it be associated to a particular individual? •The determination of blood is best made by means of a preliminary color test.
  • 25.
    8-25 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-25 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Testing for BloodTesting for Blood •A positive result from the Kastle-Meyer (phenolphthalein) color test is highly indicative of blood. Hemoglobin causes a deep pink color. •Alternatively, the luminol test is used to search out trace amounts of blood located at crime scenes. •Luminol produces light (luminescence) in a darkened area. Courtesy of C. Fanning Courtesy of C. Fanning Luminol with false positive (bleach) Courtesy of C. Fanning
  • 26.
    8-26 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  For many years, the most common test was the benzidineFor many years, the most common test was the benzidine color test, but because benzidine has been identified as acolor test, but because benzidine has been identified as a known carcinogen, its use has generally been discontinuedknown carcinogen, its use has generally been discontinued and replaced with the Kastle-Meyer test.and replaced with the Kastle-Meyer test.  Both the benzidine and the Kastle-Meyer color tests areBoth the benzidine and the Kastle-Meyer color tests are based on the observation that blood hemoglobin possessesbased on the observation that blood hemoglobin possesses peroxidase-like activity. Peroxidases are enzymes thatperoxidase-like activity. Peroxidases are enzymes that accelerate the oxidation of several classes of organicaccelerate the oxidation of several classes of organic compounds when combined with peroxides. For example,compounds when combined with peroxides. For example, when a bloodstain, phenolphthalein reagent, and hydrogenwhen a bloodstain, phenolphthalein reagent, and hydrogen peroxide are mixed together, oxidation of the hemoglobin inperoxide are mixed together, oxidation of the hemoglobin in the blood produces a deep pink color.the blood produces a deep pink color. Benzidine and Kastle-MeyerBenzidine and Kastle-Meyer
  • 27.
    8-27 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Field investigators haveField investigators have found Hemastix strips afound Hemastix strips a useful presumptive fielduseful presumptive field test for blood. Designed astest for blood. Designed as a urine dipstick test fora urine dipstick test for blood, the strip can beblood, the strip can be moistened with distilledmoistened with distilled water and placed in contactwater and placed in contact with a suspect bloodstain.with a suspect bloodstain. The appearance of a greenThe appearance of a green color indicates blood.color indicates blood. HemastixHemastix
  • 28.
    8-28 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ Another important presumptiveAnother important presumptive identification test for blood is theidentification test for blood is the luminol test. Unlike the benzidineluminol test. Unlike the benzidine and Kastle-Meyer tests, the reactionand Kastle-Meyer tests, the reaction of luminol with blood produces lightof luminol with blood produces light rather than color. By sprayingrather than color. By spraying luminol reagent onto a suspect item,luminol reagent onto a suspect item, investigators can quickly screen largeinvestigators can quickly screen large areas for bloodstains. The sprayedareas for bloodstains. The sprayed objects must be located in aobjects must be located in a darkened area while being viewed fordarkened area while being viewed for the emission of light (luminescence);the emission of light (luminescence); any bloodstains produce a faint blueany bloodstains produce a faint blue glow.glow. Luminol and BluestarLuminol and Bluestar
  • 29.
    8-29 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein {{ The identification of blood can be madeThe identification of blood can be made more specific if microcrystalline testsmore specific if microcrystalline tests are performed on the material.are performed on the material. Several tests are available; the twoSeveral tests are available; the two most popular ones are the Takayamamost popular ones are the Takayama and Teichmann tests. Both depend onand Teichmann tests. Both depend on the addition of specific chemicals tothe addition of specific chemicals to the blood to form characteristicthe blood to form characteristic crystals containing hemoglobincrystals containing hemoglobin derivatives. Crystal tests are far lessderivatives. Crystal tests are far less sensitive than color tests for bloodsensitive than color tests for blood identification and are more susceptibleidentification and are more susceptible to interference from contaminants thatto interference from contaminants that may be present in the stain.may be present in the stain. Microcrystalline TestMicrocrystalline Test
  • 30.
    8-30 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Once the stain has been characterized as blood, the serologistOnce the stain has been characterized as blood, the serologist determines whether the blood is of human or animal origin.determines whether the blood is of human or animal origin. The standard test is the precipitin test. Precipitin tests are basedThe standard test is the precipitin test. Precipitin tests are based on the fact that when animals (usually rabbits) are injected withon the fact that when animals (usually rabbits) are injected with human blood, antibodies form that react with the invadinghuman blood, antibodies form that react with the invading human blood to neutralize its presence. The investigator canhuman blood to neutralize its presence. The investigator can recover these antibodies by bleeding the animal and isolatingrecover these antibodies by bleeding the animal and isolating the blood serum, which contains antibodies that specificallythe blood serum, which contains antibodies that specifically react with human antigens. For this reason, the serum is knownreact with human antigens. For this reason, the serum is known as human antiserum. In the same manner, by injecting rabbitsas human antiserum. In the same manner, by injecting rabbits with the blood of other known animals, virtually any kind ofwith the blood of other known animals, virtually any kind of animal antiserum can be produced. Antiserums areanimal antiserum can be produced. Antiserums are commercially available for humans for human s and for acommercially available for humans for human s and for a variety of commonly encountered animals – for example, dogs,variety of commonly encountered animals – for example, dogs, cats, chickens and deer.cats, chickens and deer. Precipitin TestPrecipitin Test
  • 31.
    8-31 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Precipitin TestPrecipitin Test  Several techniques have beenSeveral techniques have been devised for performingdevised for performing precipitin tests on bloodstains.precipitin tests on bloodstains. The classic method is to layerThe classic method is to layer an extract of the bloodstain onan extract of the bloodstain on top of the human antiserum intop of the human antiserum in a capillary tube. Human blood,a capillary tube. Human blood, or for that matter, any proteinor for that matter, any protein of human origin in the extract,of human origin in the extract, reacts specifically withreacts specifically with antibodies present in theantibodies present in the antiserum, and indicated by theantiserum, and indicated by the formation of a cloudy ring orformation of a cloudy ring or band at the interface of the twoband at the interface of the two liquids.liquids.
  • 32.
    8-32 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Another method, calledAnother method, called gel diffusion,gel diffusion, takes advantagetakes advantage of the fact that antibodies and antigens diffuse orof the fact that antibodies and antigens diffuse or move toward one another on a plate coated with amove toward one another on a plate coated with a gel medium from a natural polymer called agar.gel medium from a natural polymer called agar. The extracted bloodstain and the human antiserumThe extracted bloodstain and the human antiserum are placed in separate holes opposite each other onare placed in separate holes opposite each other on the gel. If the blood is human, a line of precipitationthe gel. If the blood is human, a line of precipitation forms where the antigens and antibodies meet.forms where the antigens and antibodies meet. Gel DiffusionGel Diffusion
  • 33.
    8-33 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Gel DiffusionGel Diffusion  Similarly, the antigens andSimilarly, the antigens and antibodies can be induced toantibodies can be induced to move toward one anothermove toward one another under the influence of anunder the influence of an electrical field. In theelectrical field. In the electrophoretic method, anelectrophoretic method, an electrical potential is applied toelectrical potential is applied to the gel medium; a specificthe gel medium; a specific antigen-antibody reaction isantigen-antibody reaction is denoted by a line of precitationdenoted by a line of precitation formed between the holeformed between the hole containing the blood extractcontaining the blood extract and the hole containing theand the hole containing the human antiserum.human antiserum.
  • 34.
    8-34 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein 8-34 ©2011, 2008 Pearson Education, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein The precipitin test is very sensitive and requires only a small amount of blood for testing. Human bloodstains dried for 10 – 15 years and longer may still give a positive precipitin reaction. Even extracts of tissue from mummies four to five thousand years old have given positive reactions with this test. Furthermore, human bloodstains diluted by washing in water and left with only a faint color may still yield a positive precipitin reaction.
  • 35.
    8-35 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein  Once it has been determined that the bloodstainOnce it has been determined that the bloodstain is human, an effort must be made to associate oris human, an effort must be made to associate or disassociate the stain with a particulardisassociate the stain with a particular individual. Until the mid-1990’s, routineindividual. Until the mid-1990’s, routine characterization of bloodstains included thecharacterization of bloodstains included the determination of A-B-O types; however, thedetermination of A-B-O types; however, the widespread use of DNA profiling or typing haswidespread use of DNA profiling or typing has relegated this subject to one of historical interestrelegated this subject to one of historical interest only.only. Precipitin TestPrecipitin Test
  • 36.
    8-36 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • The precipitin test uses antisera normally derived fromThe precipitin test uses antisera normally derived from rabbits that have been injected with the blood of a knownrabbits that have been injected with the blood of a known animal to determine the species origin of a questionedanimal to determine the species origin of a questioned bloodstain.bloodstain. • Once it has been determined that the bloodstain is ofOnce it has been determined that the bloodstain is of human origin, an effort must be made to associate orhuman origin, an effort must be made to associate or dissociate the stain with a particular individual.dissociate the stain with a particular individual. • DNA analysis has allowed forensic scientists to associateDNA analysis has allowed forensic scientists to associate blood to a single individual.blood to a single individual. Recap
  • 37.
    8-37 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • Prior to the advent of DNA typing, bloodstains werePrior to the advent of DNA typing, bloodstains were linked to a source by A-B-O typing and thelinked to a source by A-B-O typing and the characterization of polymorphic blood enzymes andcharacterization of polymorphic blood enzymes and proteins.proteins. • This approach has now been supplanted by the newerThis approach has now been supplanted by the newer DNA technology.DNA technology. • DNA analysis has allowed forensic scientists to associateDNA analysis has allowed forensic scientists to associate blood and semen stains to a single individual.blood and semen stains to a single individual. A-B-O vs. DNA
  • 38.
    8-38 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • The transmission of hereditary material is accomplished byThe transmission of hereditary material is accomplished by means of microscopic units called genes, located onmeans of microscopic units called genes, located on chromosomes.chromosomes. • Alternative forms of genes that influence a givenAlternative forms of genes that influence a given characteristic (such as eye color or blood type) are known ascharacteristic (such as eye color or blood type) are known as alleles.alleles. • Paternity testing has historically involved the A-B-O bloodPaternity testing has historically involved the A-B-O blood typing system, along with blood factors other than A-B-O.typing system, along with blood factors other than A-B-O. • Currently, paternity testing has implemented DNA testCurrently, paternity testing has implemented DNA test procedures that can raise the odds of establishing paternityprocedures that can raise the odds of establishing paternity beyond 99 percent.beyond 99 percent. Heredity and PaternityHeredity and Paternity
  • 39.
    8-39 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • Many of the cases sent to a forensic laboratoryMany of the cases sent to a forensic laboratory involve sexual offenses, making it necessary toinvolve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains.examine exhibits for the presence of seminal stains. • The best way to locate and at the same timeThe best way to locate and at the same time characterize a seminal stain is to perform the acidcharacterize a seminal stain is to perform the acid phosphatase (an enzyme secreted into seminal fluid)phosphatase (an enzyme secreted into seminal fluid) color test.color test.  A purple color indicates acid phosphataseA purple color indicates acid phosphatase enzyme.enzyme. Testing for Seminal StainsTesting for Seminal Stains
  • 40.
    8-40 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein Testing for Seminal StainsTesting for Seminal Stains Semen can be unequivocally identified by either the presence of spermatozoa or of p30, a protein unique to seminal plasma. Forensic scientists can successfully link seminal material to an individual by DNA typing.
  • 41.
    8-41 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • The rape victim must undergo a medical examination asThe rape victim must undergo a medical examination as soon as possible after the assault.soon as possible after the assault. • At that time the appropriate items of physical evidenceAt that time the appropriate items of physical evidence including clothing, hairs, and vaginal and rectal swabsincluding clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination.can be collected for subsequent laboratory examination. • All outer and undergarments should be carefullyAll outer and undergarments should be carefully removed and packaged separately in paper (not plastic)removed and packaged separately in paper (not plastic) bags.bags. • Bedding, or the object upon which the assault took place,Bedding, or the object upon which the assault took place, may also be carefully collected.may also be carefully collected. Rape EvidenceRape Evidence
  • 42.
    8-42 ©2011, 2008 PearsonEducation, Inc. Upper Saddle River, NJ 07458 FORENSIC SCIENCE: An Introduction, 2nd ed. By Richard Saferstein • If a suspect is apprehended within 24 hours of the assault,If a suspect is apprehended within 24 hours of the assault, it may be possible to detect the victim’s DNA on theit may be possible to detect the victim’s DNA on the male’s underwear or on a penile swab of the suspect.male’s underwear or on a penile swab of the suspect. • Items routinely collected from the suspect include allItems routinely collected from the suspect include all clothing, pubic hair, head hair, penile swab, and a bloodclothing, pubic hair, head hair, penile swab, and a blood sample or buccal swab for DNA typing.sample or buccal swab for DNA typing. • The forceful physical contact between victim and assailantThe forceful physical contact between victim and assailant may result in a transfer of such physical evidence ofmay result in a transfer of such physical evidence of blood, semen, saliva, hairs, and fibers.blood, semen, saliva, hairs, and fibers. Rape EvidenceRape Evidence