Serology

Serology

Presumptive testing: a screening test done when you think a fluid is blood or semen. VIaible color change to the sample when a potential component of fluid is present

• Kastle- Meyer (KM)
• Acid Phosphatase (AP)

Confirmatory testing – performed after a presumptive test gives a positive result. Confirmatory tests will verify the presence of a specific fluid.

• Takayama crystal test for blood, RSID Blood Test
• Microscopic Sperm searches, RSID Semen Strip Test

Blood

• Cellular material (solid materials; 45%)
  • red blood “cells” (erythrocytes)
  • no nucleus
  • contains antigens—responsible for blood types
  • white blood cells (leucocytes)
  • platelets (thrombocytes)
• Plasma (fluid portion of blood; 55%)
  • 95% water
  • Antibodies
  • vitamins/minerals
  • 500+ proteins

Blood Typing

• An individual whose blood is type A has A antigens on his/her red blood cells.
• Type B has B antigens.
• Type AB has both A and B antigens.
• Type O has neither A nor B antigens.
• Type A blood has only anti-B antibodies and no anti-A.
• Type B blood has only anti-A and no anti-B.
• Type AB blood has neither anti-A nor anti-B.
• Type O blood has both anti-A and anti-B.

Presumptive Testing

Kastle-Meyer Test (KM)

• KM reagent added to stain
  • color change at this point: false positive
• 3% H2O2 subsequently added
• Pink color change – KM positive sample
  • Highly indicative of blood

Luminol test or Bluestar test

• used to search out trace amounts of blood located at crime scenes.
• These tests produce light (chemiluminescence) in a darkened area.
• Luminol is extremely sensitive and is capable of detecting blood that has been diluted up to 100,000 times.

Confirmatory Testing

• Takayama crystal test for blood
  • a crystalline test that is viewed using a microscope.
• RSID Blood Test

Semen

• Avg. ejaculate is 3.5mL
• Medical Conditions:
  • Oligospermia – low sperm count
  • Aspermia – no sperm
  • Vasectomy –surgical procedure that leaves the male incapable of producing sperm

Components of Semen

• spermatozoa
• Sugars
  • Sucrose
  • Fructose
  • sorbitol
• • proteins
• • inorganic ions
  • Na, K, Ca, Mg
• • buffering agents
  • Bicarbonate
  • guards against pH change in semen

Presumptive Testing

Acid Phosphatase Test

• An enzyme secreted into seminal fluid
• A purple color indicates the presence of acid phosphatase enzyme.
• Found in high concentrations in semen
• Not unique to semen
  • vaginal secretions
  • blood of males with prostate cancer
• Enzymatic activity
  • Drops significantly after 4-6 months
• Visual examination with naked eye
  • Crusty white or yellowish staining
• Alternate Light Source (ALS) examination
  • Possible semen stains will fluoresce due to bacterial growth.
  • Fresh semen stains will not fluoresce

* Semen can be specifically identified by the presence of spermatozoa

Confirmatory Testing

Christmas Tree Staining

• Red
  • aluminum sulfate + Nuclear Fast Red
  • stains DNA (nuclei)
• Green
  • picric acid + indigo carmine (PIC)
  • stains cytoplasm

RSID (Rapid Stain Identification)

• Test for presence of semenogelin

Sexual Assault Evidence

• Pubic combings
• Pubic hair controls (25)
• External genital swabs
• Vaginal swabs
• Cervix swabs
• Rectal swabs
• Swabs of body areas such as breasts
• Oral swabs
• Head hairs (25)
• Blood sample
• Buccal swab
• Fingernail scrapings
• Urine specimen
• All clothing

Saliva

Components

• Human salivary glands produce 1-1.5L/day
• 99.5% water plus electrolytes
• Mucus
• White blood cells
• Epithelial cells
• Enzymes
  • Amylase (enzyme that digests starch; α & β)
  • α amylase found in saliva and pancreas

Presumptive Testing

• Starch-iodine assays
  • Amylase overlay assay and amylase radial diffusion
  • Tests for presence of starch
  • If starch is present - will change to dark blue-black in the presence of iodine
  • False positive reactions- any substance with amylase activity (e.g. bacteria, plants, vomit)
  • Not species-specific
• Seratec® α-Amylase Test (monoclonal antibody test)
  • Positive (two lines): A line appears in the Control region, and a positive line appears within the test region.
• Phadebas Amylase Test
  • Add saliva stain to water
  • Add tablet that consists of insoluble starch bound to a blue dye
  • If amylase is present, it will break down the starch and release the blue dye

Confirmatory Testing

• RSID for saliva

Urine

• All animals must get rid of excess nitrogen from the breakdown of proteins and amino acids
• Aquatic animals get rid of nitrogen as ammonia; Birds and terrestrial animals use uric acid; Mammals use urea and expel it in urine

Presumptive testing

• Urease Test
  • Uses Whatman Filter Paper: If urea is present, then urease will catalyze the reaction of urea to ammonia and the paper will turn blue
• RSID-Urine
  • tests for a glycoprotein abundant in urine
• NOT HUMAN SPECIFIC
• No confirmatory tests for urine!

Targeted testing: Direct to DNA

• Modern DNA quantitation kits more sensitive than serology
  • Which samples have male DNA?
• Maximizes the chance of getting a DNA profile
  • For searching in CODIS
• More efficient than serology testing
  • Allows the lab to analyze evidence and obtain info necessary to search CODIS in a more timely manner

Benefits

• detect best sample(s) from kit to send for amplification
• detect presence of male DNA on samples where serology would not be an effective screening tool
  • digital penetration cases
  • sexual assaults without ejaculation
• reduce false positives (serology positive, no male profile)

DNA

Characteristics

• Genetic material that makes us who we are
• Present in almost all of your cells
• The same in all of your cells, which allows us to compare blood to saliva to skin cells
• Unique to every individual; no one else on the planet has the same DNA as you unless you have an identical twin

What part is analyzed?

• 24 different DNA locations are typically analyzed as part of a DNA profile:
  • These 24 DNA locations are know as Short Tandem Repeats (STR’s) and have no known function
  • Profiles Do Not include DNA for any visible traits such as race, eye color, hair color or diseases

Short Tandem Repeats

• Small regions along the DNA chain where a sequence (T, C, A, T) is repeated consecutively
• The number of repeats in a region can vary from person to person.
• The number of repeats are counted like “train cars.”

Samples for DNA testing

• Evidence from scene
  • Crime Scene Unit
• From victim’s body
  • Rape kits
  • Autopsy
• Exemplars (blood or mouth swab)
  • Victim
  • Suspect
  • Witnesses
  • Elimination samples

Touch DNA

• (skin cell) DNA evidence can be defined as evidence with no visible staining
  • Would likely contain DNA resulting from the transfer of epithelial cells to an object.
• Can simply touching an object leave skin cells?
  • Does not guarantee a meaningful DNA profile.

Types of DNA

• STR
  • Biological stains, hair roots, tissue
• Y STR
  • Sexual assault mixtures, body ID
• mt DNA
  • Hair shafts, body ID

What DNA does NOT do

• Forensic DNA testing does NOT provide the race of the biological fluid donor
• Forensic DNA testing does NOT provide the height, weight, hair color, birthdate, hobbies, or address of the donor
• Forensic DNA testing does NOT indicate when the stain was deposited or when the person died

Contamination

• the introduction of anything to the scene or to a piece of evidence that was not
• previously there”
• Can happen at any step after the crime has been committed
  • First responders to the scene
  • At evidence collection
  • During item sampling at the laboratory
  • At any of the DNA processing steps
• Active or “Direct” transfer: direct contact/touching of an item or surface
• Passive or “Indirect” transfer: your DNA detected on an item after not having touched it

Cross contamination

• Transfer of material from one item of evidence to another
  • (i.e. items packaged together in a bag)
• Transfer of material from an item or tool to a piece of evidence
  • (i.e. not cleaning scissors used to cut blood stains from one case to the next)
• Transfer of material on gloves from one item/case to another
  • (i.e. not changing gloves between items/cases)

Cases of Contamination

• “Phantom of Heilbronn”
  • DNA evidence from forty crimes
  • Six murders over sixteen years, dozens of high-profile thefts, and a deadly arson case
  • $400,000 bounty was placed
  • German police declared the perpetrator the “country’s most dangerous woman”
  • Cleverly evaded her sloppy criminal work and evidence left behind
  • An extensive manhunt for over 15 years despite
  • The cotton swabs used by many state police departments were found to have been contaminated before shipping by a factory worker

Mechanisms of transfer

• Direct transfer
  • Transferring cells and DNA to an object or surface after touching it
• Aerosol transfer
  • Transfer of DNA through saliva
  • Talking, shouting, coughing, sneezing, etc., over the evidence
• Indirect transfer
  • DNA on an item without having touched it
    • Secondary transfer
      • Shaking hands
      • Touching doorknob
    • Tertiary transfer
      • 1st person touches doorknob. 2nd person touches doorknob then pen

Role of Forensic Biologist

• Examine physical evidence for the presence of biological fluids or skin cells.
• Identify the fluid.
• Individualize the source of the fluid or skin cells through the generation of DNA profiles.
• Associate or link the evidence with another case or a known individual through these DNA profiles.

Sample Flow

1. Evidence Examinartion
   1. Gloves, lab coats, eye protection, masks, hair covering
   2. Cover table with paper
   3. Examine by eyes, touch, smells
   4. Pens, scissors, tissues, swabs, rulers, digital camera, microscope, Sharpies
   5. Bleach & alcohol
   6. Paperwork & notes
2. Extraction
   1. Chelex®
      1. blood, semen, saliva, hair
   2. Organic
      1. tissue, hairs
   3. M48 (QIAgen)
      1. exemplars, blood, “other” evidence
   4. SDS/Proteinase K
      1. nails
3. Quantification
   1. real-time PCR (Quantifiler Trio)
   2. Need as little as 37.5 pg (3.75-11g) of DNA to generate a DNA profile
4. Amplification
   1. Polymerase chain reaction (PCR)
   2. DNA target Loci
5. STRs/DNA typing
   1. Separation & Analysis
      1. ABI 3500xl capillary electrophoresis
      2. GeneMapper/GeneMarker software

Statistics

Statistics: the power of STRs

• Comes from combinations of loci
• Independent frequencies can be multiplied
• The 24 forensic loci yield overall frequencies which are virtually unique

CODIS

• Combined DNA Index System
• LDIS- local DNA index system—> SDIS- state DNA index system —> NDIS- national “ “
• Laws permit collection of convicted offenders
• Allows for country wide comparison of DNA evidence
• Case to case hits – combine investigation
• Case to offender matches – case potentially solved
• Missing Persons Index

Indexes of different types of samples

• Forensic (evidence) samples
• Convicted offenders/Arrestees (Depends on State rules)
• Missing persons reference samples
• Unidentified human remains