SerologySerology

Study of bodily fluids Study of bodily fluids using antigen-antibody using antigen-antibody

reactionsreactions

Blood ComponentsBlood Components

RBCRBC –– Erythrocytes ErythrocytesWBC – WBC – LeukocytesLeukocytesPlatelets – Platelets – Contains fibrin Contains fibrin which clots bloodwhich clots blood

Plasma –Plasma – Fluid portion of the Fluid portion of the un-clotted bloodun-clotted blood

SerumSerum –– Fluid that is separated Fluid that is separated from clotted bloodfrom clotted blood

Antigen – Antibody Antigen – Antibody Reactions Reactions

Antigens-Antigens- proteins on surface of cells proteins on surface of cells Antibodies- Antibodies- proteins that are produced proteins that are produced in response to the presence of foreign in response to the presence of foreign antigens (Found in the serum of blood)antigens (Found in the serum of blood)

Agglutination-Agglutination- reaction that occurs reaction that occurs when specific antibodies attach to when specific antibodies attach to antigen sites and cause RBC to clump antigen sites and cause RBC to clump togethertogether>15 blood antigen systems have been >15 blood antigen systems have been identified, including A-B-O and Rh systems identified, including A-B-O and Rh systems

( Saferstein, 2004, pg 330)( Saferstein, 2004, pg 330)

What Makes Blood What Makes Blood Different?Different?

AgglutinationAgglutination

ABO SystemABO System

Blood TypeBlood Type Antigens/RBCAntigens/RBC Antibodies/Antibodies/SerumSerum

AA AA Anti-BAnti-B

BB BB Anti-AAnti-A

ABAB ABAB Neither anti-A Neither anti-A or Bor B

OO Neither A Neither A nor Bnor B

Both anti-A Both anti-A and Band B

Blood TypingBlood Typing

Anti-A Anti-A serum + serum + bloodblood

Anti-B Anti-B Serum + Serum +

bloodblood

Antigen Antigen PresentPresent

Blood Blood TypeType

++ __

__ ++

++ ++

__ __

Immunoassay TechniquesImmunoassay Techniques

Antigen-Antibody reactions used Antigen-Antibody reactions used for the detection of Drugs in for the detection of Drugs in blood, urine and semenblood, urine and semenEMITEMIT : Enzyme-Multiplied Immunoassay : Enzyme-Multiplied Immunoassay Technique – are not totally specificTechnique – are not totally specific

Monoclonal Antibody TestsMonoclonal Antibody Tests: : antibodies are produced to react to antibodies are produced to react to ONE specific antigenONE specific antigenCurrently found in newest forensic test Currently found in newest forensic test kits kits (pg333-335)(pg333-335)

Standard Analysis of Standard Analysis of BloodBlood

Questions about crime scene Questions about crime scene blood:blood:Is the substance blood?Is the substance blood?Is the blood human?Is the blood human?Can the blood be associated with a Can the blood be associated with a particular individual?particular individual?

Is the substance blood?Is the substance blood?

Kastle-Meyer Color TestKastle-Meyer Color TestPhenolphthalein, hydrogen peroxide, Phenolphthalein, hydrogen peroxide, and suspected blood stain are mixed; and suspected blood stain are mixed; positive reaction indicated by a positive reaction indicated by a deep pink colordeep pink colorBased upon observation that hemoglobin Based upon observation that hemoglobin possesses peroxidase activity possesses peroxidase activity

This test is a presumptive or This test is a presumptive or preliminary test for blood. preliminary test for blood.

Substances other than blood may produce Substances other than blood may produce false positive results false positive results (pg 336)(pg 336)

Is the substance blood? Is the substance blood? Con’t Con’t

Hemastix ColorHemastix Color Test Test Hemastix—has been found to be very Hemastix—has been found to be very useful by field investigators—a useful by field investigators—a dipstick moistened with distilled dipstick moistened with distilled water is placed in contact with water is placed in contact with the suspect blood—a green color the suspect blood—a green color change indicates a positive result change indicates a positive result (pg 336)(pg 336)

Is the substance blood? Is the substance blood? Con’tCon’t

Luminol TestLuminol TestA test that produces light when it A test that produces light when it reacts with blood rather than colorreacts with blood rather than color

Large areas can be quickly screened Large areas can be quickly screened for blood, by spraying luminol for blood, by spraying luminol reagentreagent

““Luminol test is extremely sensitive—Luminol test is extremely sensitive—it is capable of detecting blood it is capable of detecting blood stains that have been diluted up to stains that have been diluted up to 300,000 times.” 300,000 times.”

Does not interfere with subsequent Does not interfere with subsequent DNA testing! DNA testing! (pgs 336-337)(pgs 336-337)

Is the blood human?Is the blood human?

Precipitin TestPrecipitin TestIf you inject an animal with human If you inject an animal with human blood, the animal will create blood, the animal will create antibodies that can then be recovered antibodies that can then be recovered and used to test for various human and used to test for various human antigens including human blood antigensantigens including human blood antigens

The recovered antibodies are called The recovered antibodies are called human antiserumhuman antiserum

To test for human blood, human To test for human blood, human antiserum and an unknown blood are put antiserum and an unknown blood are put together—if the blood is human a together—if the blood is human a precipitate precipitate will form.will form.

(p338)(p338)

Can the blood be associated Can the blood be associated with a particular with a particular

individual?individual? A-B-O types and enzymesA-B-O types and enzymes

Used until the Used until the mid 1990’smid 1990’s, , characterization of bloodstains included characterization of bloodstains included determining A-B-O types and polymorphic determining A-B-O types and polymorphic enzyme markers such as PGM enzyme markers such as PGM (phosphoglucomutase)(phosphoglucomutase)

These tests added statistical information These tests added statistical information with which to reduce the number of with which to reduce the number of possible sources of the bloodstainpossible sources of the bloodstain

Because antigens, and enzymes occur Because antigens, and enzymes occur independently of one another, the independently of one another, the probability of a dried blood stain having probability of a dried blood stain having a particular combination of these factors a particular combination of these factors is determined by the product of their is determined by the product of their distribution in the population distribution in the population (pg 339)(pg 339)

Can the blood be associated Can the blood be associated with a particular with a particular individual? Con’tindividual? Con’t

Statistical Example:Statistical Example:Blood stain found to be of type A— Blood stain found to be of type A— corresponds to 42% percent of corresponds to 42% percent of population population

Stain contains PGM-1—corresponds to Stain contains PGM-1—corresponds to 58% of population58% of populationThe combined effect of just these two The combined effect of just these two factors, using the product rule is 42% x factors, using the product rule is 42% x 58% or 24 %58% or 24 %

Clearly the more factors, the smaller the Clearly the more factors, the smaller the frequency of occurrence in the populationfrequency of occurrence in the population

Can the blood be associated Can the blood be associated with a particular with a particular individual? Cont….individual? Cont….

DNA AnalysisDNA Analysis Since the 1990’s DNA analysis has Since the 1990’s DNA analysis has replaced forensic reliance on replaced forensic reliance on blood factors for the routine blood factors for the routine characterization of bloodstains characterization of bloodstains

DNA analysis gives extremely DNA analysis gives extremely accurate individualization accurate individualization results. results.

Stain Patterns of BloodStain Patterns of Blood

The location, distribution and The location, distribution and appearance of bloodstains and appearance of bloodstains and spatters can be used to spatters can be used to interpret and reconstruct the interpret and reconstruct the events that must have occurred events that must have occurred to produce the bleeding. to produce the bleeding. (pg. 342)(pg. 342)

Stain Pattern AnalysisStain Pattern AnalysisGeneral Conclusions General Conclusions

Surface textureSurface texture: The harder and : The harder and less porous the surface, the less porous the surface, the less spatter results.less spatter results.

Direction of TravelDirection of Travel: The : The direction of travel of blood can direction of travel of blood can be discerned by the stain’s be discerned by the stain’s shape — shape — the pointed end always the pointed end always faces its direction of travel faces its direction of travel (pg 342)(pg 342)

Stain Pattern Analysis Stain Pattern Analysis General Conclusions Cont…General Conclusions Cont…

Impact Angle:Impact Angle: A drop of blood striking a A drop of blood striking a surface at a right angle gives rise to a surface at a right angle gives rise to a nearly circular stain; as the angle nearly circular stain; as the angle decreases, the stain becomes more decreases, the stain becomes more elongated in shapeelongated in shape

Origin of Blood Spatter:Origin of Blood Spatter: Can be Can be determined by establishing lines through determined by establishing lines through the long axis of several individual blood the long axis of several individual blood stains. The intersection of the lines stains. The intersection of the lines represents the point from which the blood represents the point from which the blood emanated. emanated. (pg 342)(pg 342)

http://www.bloodspatter.com/BPATutorial.hhttp://www.bloodspatter.com/BPATutorial.htmtm

Principles of HeredityPrinciples of Heredity

The gene is the basic unit of The gene is the basic unit of heredity. They found on heredity. They found on chromosomes. All human cells chromosomes. All human cells contain 46 chromosomes (23 pairs). contain 46 chromosomes (23 pairs). Only exception: reproductive cellsOnly exception: reproductive cells

During fertilization, a sperm and During fertilization, a sperm and egg combine so each contributes egg combine so each contributes chromosomes to form the new cell chromosomes to form the new cell (zygote)(zygote)

Principles of Heredity Principles of Heredity Cont..Cont..

Inheritance RulesInheritance RulesA gene pair of two similar genes—A gene pair of two similar genes—is is homozygoushomozygous—no problem —no problem determining a person’s outward determining a person’s outward characteristics characteristics

A gene pair of two different genesA gene pair of two different genes—is —is heterozygousheterozygous—one gene is —one gene is dominantdominant, the other , the other recessiverecessive——person’s outward characteristics person’s outward characteristics depend on the dominant genedepend on the dominant gene

Paternity TestingPaternity Testing

Many cases of disputed Many cases of disputed paternity can be resolved when paternity can be resolved when suspected parents and offspring suspected parents and offspring are related according to their are related according to their blood group systemsblood group systems

Type O blood is considered a Type O blood is considered a recessive gene so……recessive gene so……

Paternity ExamplePaternity Example

Father’s Genotype

O O

Mother’s

Genotype

A

B

AO AO

BO BO

If the Child in question is type AB If the Child in question is type AB would the suspect father be cleared? would the suspect father be cleared?

Paternity Testing Con’tPaternity Testing Con’t

Routine paternity testing Routine paternity testing involves characterizing many involves characterizing many blood factors other than A-B-O. blood factors other than A-B-O. Using these other factors the Using these other factors the probability of establishing probability of establishing paternity becomes better than paternity becomes better than 90%. Once DNA analysis is done 90%. Once DNA analysis is done the odds become better than the odds become better than 99%. 99%. (pg 348)(pg 348)

Forensic Characterization Forensic Characterization of Semenof Semen

A large number of cases involve sexual A large number of cases involve sexual offenses, making it necessary to offenses, making it necessary to examine exhibits for the presence of examine exhibits for the presence of seminal stains. seminal stains.

The forensic examination of semen is a The forensic examination of semen is a two-step process:two-step process:

1.1. Semen must be locatedSemen must be located

2.2. Tests must be performed to prove its Tests must be performed to prove its identity—it may even be tested for blood identity—it may even be tested for blood typetype

Note: Semen is checked for blood type because Note: Semen is checked for blood type because many individuals are many individuals are secretors secretors

SecretorsSecretors

80% of individuals are classified as 80% of individuals are classified as secretorssecretors

This means that their blood-type This means that their blood-type antigens are also found in high antigens are also found in high concentrations in most body fluidsconcentrations in most body fluids

Ex- Saliva, semen, vaginal Ex- Saliva, semen, vaginal secretions, and gastric juicessecretions, and gastric juices

**Saliva & semen have higher **Saliva & semen have higher concentrations of A & B antigens concentrations of A & B antigens than blood!!than blood!!

Semen Location and Semen Location and IdentificationIdentification

There are two methods of There are two methods of locating semen:locating semen:1.1. Semen is often Semen is often readily visiblereadily visible on on

a fabric because it has a stiff a fabric because it has a stiff crusty appearance- unreliablecrusty appearance- unreliable

2.2. The The acid phosphatase color testacid phosphatase color test can locate and at the same time can locate and at the same time identify semen. identify semen.

Acid Phosphatase Color Acid Phosphatase Color TestTest

Acid phosphatase is an enzyme Acid phosphatase is an enzyme secreted by the prostate gland into secreted by the prostate gland into seminal fluid that is in far greater seminal fluid that is in far greater concentrations than those found in concentrations than those found in any other body fluids.any other body fluids.

Its presence can be detected using Its presence can be detected using different tests:different tests:1. Contact with a special acidic 1. Contact with a special acidic solution and Fast Blue B dye shows a solution and Fast Blue B dye shows a purple change purple change

2. Contact with a solution of MUP 2. Contact with a solution of MUP fluoresces under UV light. fluoresces under UV light. (p.349)(p.349)

Acid Phosphatase Test, Acid Phosphatase Test, Con’tCon’t

Large areas or fabrics can be quickly Large areas or fabrics can be quickly searched by moistening filter paper searched by moistening filter paper with the detector solutions and with the detector solutions and rubbing over the suspect arearubbing over the suspect area

Some substances will give a false-Some substances will give a false-positive result (fruit juice, positive result (fruit juice, contraceptive creams, etc), but none contraceptive creams, etc), but none of these substances will react with of these substances will react with the speed of seminal fluid. the speed of seminal fluid. Note: A reaction of less than 30 seconds Note: A reaction of less than 30 seconds is a strong indication of semen. is a strong indication of semen. (p 349)(p 349)

Microscopic Examination of Microscopic Examination of SemenSemen

Rapid stirring of a fabric-Rapid stirring of a fabric-stain-water mixture will stain-water mixture will transfer a small amount of transfer a small amount of sperm into the water. A drop is sperm into the water. A drop is then taken, dried, stained and then taken, dried, stained and looked at under the microscope.looked at under the microscope.

The presence of sperm indicates The presence of sperm indicates the presence of semen.the presence of semen.

Microscopic ProblemsMicroscopic Problems

Locating sperm with the microscope is Locating sperm with the microscope is not that easy for three reasons:not that easy for three reasons: 1. Sperm are very brittle when dried and 1. Sperm are very brittle when dried and easily disintegrate. They are also hard easily disintegrate. They are also hard to remove from cloth material.to remove from cloth material.

2. Sexual crimes may involve males who 2. Sexual crimes may involve males who have an abnormally low sperm count, have an abnormally low sperm count, condition called condition called oligospermia.oligospermia.

3. Males who have no sperm in their 3. Males who have no sperm in their seminal fluid, condition called seminal fluid, condition called aspermiaaspermia——often due to increasing numbers of often due to increasing numbers of vasectomies. vasectomies.

Detecting Semen, Cont…Detecting Semen, Cont…

A positive result for acid A positive result for acid phosphatase and a negative phosphatase and a negative result for semen give ambiguous result for semen give ambiguous results—In this case, p-30 or results—In this case, p-30 or Prostate Specific Antigen (PSA) Prostate Specific Antigen (PSA) is used to detect seminal is used to detect seminal plasma – Immunoassay testsplasma – Immunoassay tests

p30 Prostate Specific p30 Prostate Specific Antigen TestAntigen Test

Formation of a visible line Formation of a visible line between the wells of anti p-30 between the wells of anti p-30 and semen extract–proves the and semen extract–proves the stain is seminalstain is seminal

Collection of Rape EvidenceCollection of Rape EvidenceVictimVictim

Outer and undergarments are placed in Outer and undergarments are placed in separate paper bags, while victim stands separate paper bags, while victim stands on a piece of paper. The paper is then on a piece of paper. The paper is then folded and collected.folded and collected.

Bedding or the object upon which assault Bedding or the object upon which assault took place should be submitted to the took place should be submitted to the lab. lab.

Rape victim must be subjected to a Rape victim must be subjected to a medical examination Collected items medical examination Collected items include pubic hairs, vaginal swabs, include pubic hairs, vaginal swabs, blood, fingernail scrapings, clothing and blood, fingernail scrapings, clothing and urine urine (p 352)(p 352)

Rape SuspectRape Suspect

Suspect must also undergo an Suspect must also undergo an exam. Routine items collected exam. Routine items collected include clothing, hairs, penile include clothing, hairs, penile swab and blood samplesswab and blood samples

Timeline Timeline

Living or motile sperm may survive Living or motile sperm may survive 4 to six hours in the vagina of a 4 to six hours in the vagina of a living person. living person.

Non-motile sperm may be found in a Non-motile sperm may be found in a living female for up to three days living female for up to three days and occasionally up to six daysand occasionally up to six days

Intact sperm (sperm with tails) Intact sperm (sperm with tails) are not normally found 16 hours are not normally found 16 hours but have been found as late as 72 but have been found as late as 72 hours after intercourse. hours after intercourse.

Important Considerations Important Considerations Based Upon TimelineBased Upon Timeline

How long ago was the person How long ago was the person raped?raped?

When did voluntary sexual When did voluntary sexual activity last occur prior to activity last occur prior to the sexual assault?the sexual assault?Blood is taken from any consensual Blood is taken from any consensual partner having sex with the victim partner having sex with the victim within 72 hours of the assault. within 72 hours of the assault.

What are these?What are these?