Chapter 8 FORENSIC SEROLOGY Nature of Blood Blood is a

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FORENSIC SEROLOGY

• Chapter 8

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Nature of Blood

• Blood is a highly complex mixture of cells,
  enzymes, proteins, and inorganic substances.
• Plasma, is the fluid portion of blood and
  composed principally of water.
• Red blood cells (erythrocytes), white blood cells
  (leukocytes), and platelets are the solid
  materials suspended in plasma.
• Antigens, (proteins) are located on the surface
  of red blood cells and are responsible for
  blood-type characteristics.

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Serology-study of biological fluids involving
specific antigen and serum antibody reactions

• 1901-Karl Landsteiner-ABO blood type system
• Blood types based on presence of antigens on
  surface of red blood cells (erythrocytes)

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Blood Typing

• 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.
• Rh factor is determined by the presence of
  another antigen, the D antigen.
• People having the D antigen are Rh positive
  those not having the antigen are Rh negative.
• For every antigen there is a specific antibody
  that will react with it to form clumps known as
  agglutination.
• Thus, if serum containing anti-B is added to red
  blood cells carrying B antigen, they will
  immediately react.

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Antigens

• Proteins that stimulate production of antibodies
• On surface of erythrocytes
• Types- A and B
• Antibodies-produced against antigens not present
• Ex. Type A blood has A antigens, but B antibodies

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Blood Types
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andsteiner/
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Frequency of Blood Types
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Blood Typing

• Blood typing requires two antiserums
• anti-A and anti-B. By inserting a droplet of
• these antiserums in samples of blood, one
• can determine which samples maintain a
• normal appearance and which samples
• become clotted, or agglutinated, under
• microscopic examination. Type-A blood will
• be agglutinated by anti-A serum Type-B
• blood will be agglutinated by anti-B serum
• Type-AB blood by both and Type-O blood by
• neither.

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Blood Factors

• If mixed-blood agglutinates (solidifies) -death
• Rh factor (antigen D)
• More than 100 blood factors

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Blood

• Cells-
• Red blood cells (Erythrocytes)
• Platelets (Thrombocytes)
• White blood cells (Leukocytes)
• 2. Enzymes
• 3. Proteins
• 4. Fluid
• 5. Inorganic substances (gases)

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Blood Plasma

• Liquid part of blood
• Serum (liquid that separates from blood) and
  proteins in water
• 55 of blood volume
• Fibrin-blood clotting protein

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Erythrocytes

• Red blood cells
• Transport oxygen and carbon dioxide
• Has hemoglobin-blood protein carries oxygen in
  blood
• Most numerous blood cells

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Leukocytes (White Blood Cells)

• Part of Immune System
• Fight infections

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Platelets

• Thrombocytes
• Involved in blood clotting

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Antiserum

• Serum that contains antibodies (proteins that
  destroy specific antigens)
• Used in determining blood types
• If antiserum causes agglutination, then the blood
  type can be determined

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Antigen-Antibody Concepts

• Basic concepts Antigens
• Antigens induce antibody formation when
• introduced into a foreign host
• Antibody-antigen Interaction Antigens and
• antibodies exhibit affinity and binding
• behaviors in solution (lock and key concept)
• Binding of antigens and antibodies leads to
• elimination of the foreign substance.

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Serology

• 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.
• The concept of specific antigenantibody
  reactions has been applied to immunoassay
  techniques for the detection of drugs of abuse in
  blood and urine.

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Figure 81  
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Immunoassay

• 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
  subjects urine.
• Other immunoassay procedures are also available,
  such as radioimmunoassay (RIA), which uses drugs
  labeled with radioactive tags.

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Immunoassay Concepts

• Basic concepts Immunoassay
• Immunoassays are a diverse group of analytical
• techniques based on specific antibody/antigen
• interactions producing a measurable signal that
  can
• be related to the concentration of a compound in
• solution
• Applications in almost every area of the
  laboratory
• this presentation will be limited to drugs of
  abuse
• assays and applications
• Antibodies may be monoclonal or polyclonal and
  may
• be directed towards very specific drug molecules
  or
• an entire drug class

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Antigen-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.
• 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.

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Immunoassay Techniques
Presumptive test for detection of drugs in blood
and urine Antibodies reacting with drugs dont
occur naturally Produced in animals by combining
drug with a protein and injecting into
animal Drug-protein complex acts as antigen
stimulating animal to produce antibodies The
recovered blood serum of animal contains specific
antibodies to the drug-This is human antiserum
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Immunoassays and Forensic Science

• Forensic toxicology encompasses the determination
  of the presence and concentration of drugs (and
  other substances) and their metabolites in
  physiological fluids and organs and the
  interpretation of these findings as they may
  impact on legal issues. These include medical
  examiner investigations, driving under the
  influence and other transportation accident
  investigations, workplace pre-employment, random
  and for-cause drug testing and judicial
  monitoring of arrestees and parolees.
• For the most part, forensic toxicologists use
  commercial immunoassays directed primarily
  towards abused drugs. Commercial immunoassays
  developed for therapeutic monitoring of other
  drugs, veterinary drugs and pesticides, as well
  as immunoassays developed in research
  laboratories for specialized studies, may find a
  role in the forensic toxicology laboratory for
  specialized cases.

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EMIT- Enzyme Multiplied Immunoassay
Technique-drug screening in urine and blood-add
antibodies to blood or urine, then add chemically
labeled drug- will compete with drugs in urine or
blood (if present) for antibody binding sites.
The number of remaining unbound labeled
drug-proportional to drugs concentration in
urine. ex. Methadone-add methadone antibodies and
chemically-labeled methadone to urine- ex. THC
tetrahydrocannabinol- metabolite-THC-9-carboxylic
acid-antibodies are prepared against this
metabolite-one-millionth of a gram-can detect up
to 10 days after use
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Steps required to produce monoclonal antibodies
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Polyclonal vs. Monoclonal Antibodies

• Polyclonal antibodies-series of antibodies which
  can attack some site on antigen-vary in
  specificity and binding abilities
• Monoclonal antibodies-identical antibodies that
  interact with one site on an antigen

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Monoclonal antibodies

• Immunoassay tests for abused drugs made with
  monoclonal antibodies

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Forensics of Blood

• The criminalist must answer the following three
  questions when examining suspected bloodstains
• 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?
• Question 1 can be answered by a color test.

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Color Tests

• The Kastle-Meyer color test uses phenolphthalein
  to detect blood.
• Hemoglobin causes a deep pink color.
• Luminol test can detect blood diluted up to
  300,000 times.
• Produces blue glow (luminescence) in a darkened
  area.
• Microcrystalline tests, Takayama and Teichmann,
  uses chemicals that form crystals if blood is
  present. Not very sensitive. Easily contaminated.

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The Tests

• To answer Question 2
• Precipitin test uses antibodies from rabbits that
  have been injected with the blood of a known
  animal to determine the species of a questioned
  bloodstain.
• Then Question 3 must be answered if the
  bloodstain is human, it must be matched to the
  crime scene, either the victim or suspect(s).
• DNA analysis has allowed forensic scientists to
  match blood to a single individual.
• Before 1990s, ABO blood typing could only be
  used to eliminate a suspect, but could not be
  used to convict them.

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Bloodstain Analysis Blood or not?

• In order to detect invisible bloodstains,
• forensic investigators use the luminol test.
• Luminol, a chemical sprayed on carpets
• and furniture, reveals a slightly
• phosphorescent light in the dark where
• bloodstains (and other stains) are present.
• Long-dried blood has a tendency to
• crystallize, or can be made to crystallize
• with various saline-acid mixtures. The
• names of various crystal tests are the
• Teichman test , the Takayama test , and
• Wagenhaar test. 

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Bloodstain AnalysisAnimal Blood?

• To determine whether blood at the crime
• scene originated from an animal, forensic
• investigators use antiserum or gel tests.
• Establishing whether or not blood is animal
• blood is significant because any possibility
• of an injury to the household pet, caused by
• a perpetrator or another animal, must be
• eliminated. Pets generally spread human
• bloodstains throughout the crime scene,
• but the pet can be a victim, perpetrator, or
• witness (through the cross transfer of
• evidence between the animals DNA and the
• perpetrator). Veterinary forensics may be a
• necessary unit if pets are involved in the
• crime.

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Bloodstain AnalysisAnimal Blood?

• To determine whether blood is animal or
• human in origin, the precipitin test is
• conducted. This process involves injecting
• an animal, usually a rabbit, with human
• blood. The rabbit's body creates
• anti-human antibodies, which are then
• extracted from the rabbit's serum. If this
• antiserum is then placed on a sample from
• the crime scene, and the blood displays
• clotting, the forensic investigator can
• conclude that the blood is human blood.
• The same procedure of creating and
• extracting antiserum can be applied to
• every known animal.

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Blood Crime SceneWet vs. Dry Blood

• Wet blood is more significant than dried
• blood because the forensic scientist can
• perform more tests in order to gain insight
• to the happenings of the crime. For
• example, alcohol and drug content can be
• determined from wet blood only. Blood
• begins to dry after three to five minutes of
• exposure to air. As it dries, it changes color
• from a deep red towards brown and black.
• Blood can be categorized into pools, drops,
• smears, or crusts.