Use of Immunology Techniques in

Crime Scene Analysis

by Forensic Scientist
Nichole Kellerman
Walter Johnson High School
Bethesda, Maryland

High School Teachers Research Program

Funded by the American Association of Immunologists
 Lesson 1-4 Objectives:
Review how each human has unique characteristic traits
found in their DNA as genes.
Describe how Forensic Technicians use these principles to
identify and exonerate suspects identified by criminal
investigations.
Obtain hair samples to extract DNA.
Perform DNA extraction techniques.
Perform polymerase chain reaction (PCR).
Analyze samples with gel electrophoresis to see if PCR
was successful.
 DNA Analysis
Each human has unique characteristic traits found in their DNA as genes.
Each cell in your body has DNA--the exception being RBC’s which mature and
lose their nucleus over time.
Three types of forensic analysis of DNA are conducted:
• Nuclear DNA
• Y chromosomal DNA
– http://projects.nfstc.org/fse/01/01-08c.html
• Mitochondrial DNA
– http://projects.nfstc.org/fse/01/01-08b.html
DNA analysis is divided into four parts:
• Extraction
• Amplification
• Detection
• Interpretation
 DNA Analysis: Extraction
DNA Extraction:
– Lyse cells: Sonicating, bead beating the sample, or
vortexing the sample with phenol . . . and then adding
SDS, a detergent, helps to remove the lipid membranes;
are effective ways for breaking apart cell membranes to
release DNA from the cell.
– Protease is used to degrade proteins associated with the
DNA or other cellular proteins. After ammonium or
sodium acetate is added to the container, a precipitate
of the cells proteins will form at the bottom of the
container. Then phenol-chloroform is added, and the
tube is vortexed. Lastly, the conical tube is centrifuged.
• The proteins will remain in the organic phase, and
this makes it easier to be pipetted out of the
container.
• The DNA will be found at the interphase between the http://quicktubes.com/english/company/business-
development/looxster.html

two phases.
 DNA Analysis: Extraction
Add cold ethanol or isopropanol into the tube and then centrifuge.
DNA will not dissolve in alcohol, so it will be seen clearly in the
solution. The alcohol also acts like a washing agent, it will remove
the salt that was added to the solution and keep the DNA separate
from everything else.

Wash the resultant DNA pellet with cold alcohol again and
centrifuge the tube again to rinse the DNA a second time. The
precipitated pellet is the DNA. http://serc.carleton.edu/details/
images/7864.html

Pour the alcohol off the DNA pellet. Then, re-suspend the DNA in
Tris (TE) Buffer.

In order to confirm the presence of DNA, run samples in a gel. Use

gel electrophoresis containing ethidium bromide or another
fluorescent dye that reacts with the DNA. Place the gel under a UVhttp://serc.carleton.edu/details/
light to illuminate the DNA samples. images/7865.html
 DNA Analysis: Extraction

If a sexual assault case involves processing DNA

from a stain of semen, additional steps are required
to separate sperm cells from the other body cells
contained within the stain. The first portion of the
sample is composed of sperm DNA. The other
portion of the sample will contain other cells. This
technique makes it possible to separate male and
female DNA.

http://projects.nfstc.org/fse/01/01-09b.html
 DNA Analysis: Amplification
In Forensics most DNA is found in residues or stains left behind. The
trace amounts of DNA can be amplified for further analysis.

Polymerase chain reaction (PCR) is a method that generates multiple

copies of DNA segments or genes from a sample. Under controlled
conditions, small segments of DNA are created by enzymes called
DNA polymerases. The polymerases add complimentary DNA
nucleotides to the existing DNA strand called the DNA template.

Smaller fragments of DNA called primers are used by the polymerase

to start the process of amplifying the DNA. Primers are usually small,
manmade pieces of DNA called oligomers. The primers are usually
15-20 nucleotide bases long.
 DNA Analysis: Amplification
During PCR, the DNA being sequenced is:
– Denatured: It is heated, and the double strand of DNA is
separate.
– Annealed: The DNA is cooled. This allows the free floating
primer sequences to bind to the DNA template strands.
– Extended: The Taq polymerase attaches to the primer and
uses the free floating nucleotides (ATP, CTP, GTP, TTP)
to replicate the DNA sequence from the DNA template.
– The number of PCR cycles (repeats) run is based on the
amount of DNA desired.
 DNA Analysis: Detection
PCR is an invaluable tool in law
enforcement as a step in the process of
DNA fingerprinting.

Detection is an automated process where

alleles for each gene location or locus are
determined. The combinations of alleles
at each locus establishes a genetic profile
described by a series of letters or
numbers.

Each sample is run through gel

electrophoresis to determine a DNA
fingerprint, a pattern of DNA fragments
that are unique to each individual. http://www.2classnotes.com/digital_notes.asp?p=DNA_Fingerprinting
DNA Analysis: Interpretation

http://projects.nfstc.org/fse/01/01-12b.html
DNA Analysis: Interpretation

http://projects.nfstc.org/fse/01/01-13b.html
 Practical Applications of DNA
Fingerprinting
1. Solving Cases of Disputed Paternity and Maternity: Because a
person inherits his or her VNTRs from his or her parents, VNTR
patterns can be used to establish paternity and maternity.

2. Criminal Identification and Forensics: DNA isolated from blood,

hair, skin cells, or other genetic evidence left at the scene of a crime
can be compared through VNTR patterns with the DNA of a criminal
suspect to determine guilt or innocence.

3. Personal Identification: The notion of using DNA fingerprints as a

sort of genetic bar code to identify individuals has been discussed, but
this is not likely to happen anytime in the near future. The technology
required to isolate, keep on file, and then analyze millions of very
specified VNTR patterns is both expensive and impractical.
 Forensic Technicians use immunology
principles to identify and exonerate suspects
identified by criminal investigations.
CSI’s collect specimens left behind by the suspect or
victim at a crime scene.
– body fluids: blood or semen
– other tissues: hair or bone

Immunological Tests are used by CSI’s in the detection

and identification of crime scene samples. They
perform presumptive test or screening tests at the
crime scene and at the lab further analysis and
confirmatory tests are completed.

DNA Typing is used to identify the collected sample

against a reference sample from a known source. The
reference sample could be taken from a potential
suspect or a known victim. The goal is to identify the
source as belonging to the suspect and not to the
http://www.nfstc.org/pdi/Subject02/
victim. DNA Typing can also exclude a person from a pdi_s02_m02_01.htm
list of potential suspects.
Explain the immunology behind confirmatory test
when identifying bodily fluid

It is important to determine very quickly if fluid residues

are blood.

Presumptive tests use a chemical reaction, linked to a

color indicator, to screen the fluid sample as blood. It
does not identify the origin of the blood as human or
other.

A confirmatory test is needed to identify the specific

body fluid as being human blood.
 Serology
The branch of laboratory medicine that studies blood serum for evidence of infection
by evaluating antibody reactions in vitro.

Blood is a fluid (suspension of cells) containing 3 types of materials:

• Salts: Na+, K+, & Cl-
• Organic chemicals: glucose, hormones, and vitamins
• Proteins

Blood contains 3 cellular components:

• RBC’s or erythrocytes:
– Mature circulating RBC are enucleated (lose their nucleus to
increase oxygen binding ability, do not contain DNA)
• Platelets or thrombocytes
– Contain a nucleus with DNA
• WBC’s or leukocytes
– Lymphocytes are the WBC responsible for antibody production
 Serology
The branch of laboratory medicine that studies blood serum for evidence of infection
by evaluating antibody reactions in vitro.

Serum is the fluid portion of clotted blood:

• The fluid portion of the unclotted blood is called plasma.
Blood clots through the conversion of a dissolved protein,
fibrinogen, to a precipitated polymer called fibrin.
• Fibrin acts like a fisherman’s net, trapping platelets to form
a clot.
• Serum can be separated by electrophoresis into albumin
and globulin.
– Albumin preserves blood volume by regulating osmotic
pressure.
– Globulin consists of many different proteins.
 Screening tests are presumptive tests

Depend on the peroxidase activity of hemoglobin, a protein within the blood

that binds oxygen.
Most tests depend on the oxidation, or loss of electron, of colorless solution
which is reduced, gains an electron.
Many of these solutions are conjugated to a known or suspected carcinogen.
Screening tests are not specific for blood because other organisms like fruit
contain peroxidase, or it can be found on the surface of objects.
Presumptive tests can also be used to identify other body fluids: saliva, semen,
vaginal fluid. . .
Preliminary tests are less time consuming, but are not definitive. Confirmatory
tests are discriminating test, because they report the origin of the fluid or body
tissue with a high degree of scientific certainty. They are usually performed
after presumptive test because they require more time.

http://projects.nfstc.org/fse/01/01-07.html
 Immunology behind confirmatory test
used by CSI’s to identify drugs

Presumptive or Screening tests:

– Determine the possible presence of controlled
substances.
– Classify these controlled substances into general
categories:
• Opium alkaloids, synthetic opiates, cocaine, indole
alkaloids, benzodiazepines, barbiturates, sedatives,
hypnotic, anesthetics, marijuana, and
phenalkylamines
Confirmatory Tests:
– Identify the identity of the controlled substance .
 Screening Process
Examine evidence.
Determine the location of biological evidence.
Methods used to locate stains are dependent on
the type of body fluid/tissue present.
– Simplest form is a swab
– Little effort is required to locate the stain on a swab
– Complicated evidence collection occurs with: blood
on a high surface, like a window, stains on clothing,
bed sheets, blankets, and carpet
– These types of evidence will require more time
Many times, there will be multiple types of
evidence at the crime scene. The screening
process may simply be seen with visual
examination, or it may require microscopes, and
alternative light sources or chemical tests.
http://projects.nfstc.org/fse/01/01-05.html
 Confirmatory Tests
Confirm the residue left behind at the crime scene
contains blood.
– Takayama test also known as the hemochromgen test:
• The oldest confirmatory test of blood detects the presence of
hemoglobin or derivatives crystals of hemoglobin. In this test,
ferrous iron within hemoglobin reacts with pyridine. If red feathery
crystals are present, the residue contains blood.
– Teichman reagent:
• In this reaction, hemoglobin is converted to hemin, and then, the
halides reacts with the hemin to form brownish-yellow rhomboid
crystals if the substance contains blood.
Identifying Blood as being Human Blood

The next step will confirm that blood belongs to a

human.
– A precipitin reaction with antibodies to blood serum is used
to determine that the sample is of human origin.
– It is an anti-human serum, an antiserum to human serum.
– In blood, serum are the proteins albumin and globulins.

– The Kastle Meyer test is a confirmatory test for human

blood.
• http://www.youtube.com/watch?v=6Ex0Fd_PDhU
 Describe the basic components of the
Immune system

The lymph node contains numerous specialized

structures. T cells concentrate in the paracortex, B cells in
and around the germinal centers, and plasma cells in the
medulla.
Immune cells and foreign particles enter the lymph nodes
via incoming lymphatic vessels or the lymph nodes’ tiny
blood vessels.

http://www.niaid.nih.gov/topics/immunesystem/Pages/structure.aspx
Investigate the crime scene

Use the scientific method to answer these questions:

– What would you consider evidence?

– How would you collect, store, and document the

evidence recovered?

– What questions need to be answered to solve the

crime?
 Lesson 2-5 Objectives:

Obtain hair samples to extract DNA.

Perform DNA extraction techniques.

Perform polymerase chain reaction.

Analyze samples with gel electrophoresis to see if PCR

was successful.
Polymerase Chain Reaction
(PCR)
What happens
when cells
copy their own
DNA?

This is the
process
mimicked
by PCR.
http://users.ugent.be/~avierstr/principles/pcr.html

http://www.dnalc.org/ddnalc/resources/pcr.html
 Developed by Dr. Kary
Mullis (1985)
“Molecular Photocopying”
Allows scientists to make millions of copies of a selected stretch of
DNA form within the total genomic DNA
Uses enzymatic amplification much like DNA replication, just at
high-speed
Does not require splicing out the selected stretch, creating a
vector, and cloning it in a bacterial or a yeast culture
 The Materials
DNA from a crime scene, from
remains ,or from a suspected illegally
poached animal (template DNA)
Pair of DNA primers that are
approximately 20 nucleotides long and
flank the target region to be amplified
Heat resistant DNA polymerase (Taq)
from the thermophilic bacterium
Thermus aquaticus
Magnesium (cofactor)
Four deoxyribonucleoside
triphosphates (dNTPs) http://www.bio.davidson.edu/Courses/Molbio/
MolStudents/spring2002/Robinson/Isocitrate-main-
page.html
 The Protocol
1. Denaturing
Heat the mixture to 94oC to separate the
template DNA into 2 strands (break
hydrogen bonds).
2. Annealing
Cool the mixture to 65oC so that the
primers anneal (stick) to the template
DNA.
Primers anneal before the DNA
template strands come back together,
because they are short and present in
excess.
3. Extending
Heat the mixture to 72oC so that DNA
polymerase can synthesize
complementary strands from the
primers.
http://www.hhmi.org/biointeractive/dna/DNAi_PCR.html

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
 The Product
Each cycle takes 2 minutes
and doubles the number of
template DNA molecules.

25 cycles takes less than

an hour and should
produce 1,000,000 copies
of template DNA
molecules.

How many copies of http://pathmicro.med.sc.edu/pcr/PCR_Reaction_graph2.htm

template DNA would you

have after 10 cycles of
PCR? After 50 cycles?
 The Machine
Once upon a time, scientists
had to move reaction tubes
among 3 water baths of
different temperatures
according to a strict timing
routine.
Now, PCR is automated
using a thermal cycler.
– A computer controller raises
and lowers the temperature of
a heat block so that all tubes
stay in one place, and the http://www.labsupply.com.hk/Thermal%20Cycler%20001.htm

scientist can go out to lunch.

 RT-PCR
First, an RNA strand is reverse
transcribed into its DNA complement
(complementary DNA or cDNA) using
the enzyme reverse transcriptase.
Then, the resulting cDNA is amplified
using traditional PCR.

http://archive.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Campbell/rt_pcr.html
Student Samples Homozygous for Heterozygous for Homozygous – no
the insert (+,+) the insert (+,-) insertion (-,-)
1

10

11

12

Totals
 Lesson 6-7 Objectives:

Identify unknown white substances by creating a standard

of color identification for known white substances and
comparing these standards to the 5 unknowns.

Perform presumptive test or screening tests at the

crime scene and at the lab to further analyze and confirm
findings.
Testing with Immunoassays
Immunoassays are used by CSI’s to
analyze specimens for the presence of
drugs.

CSI’s may use these tests at the crime

scene or while collecting evidence.

Forensic Technicians may also use these

procedures in a laboratory.
37
Testing with Immunoassays

CSI’s use immunoassays in the field because they are fast,

reliable, and consistently show a color to indicate the
presence or absence of drugs, alcohol, or presence of
bodily fluid at a crime scene.

In this investigation, a simple color indicator test will be

used to demonstrate how colors indicate the presence of
specific drugs.

38
 Procedure:
Take a wax pencil, and write on the lid of the cell
culture container these solutions. Abbreviations for
each solution can be written instead of the entire
name.
– Benedicts Solution
– Fe(NO3)3 3.0 M
– Hydrochloric acid 3.0 M
– Iodine
– Universal Indicator Color
– Water Solubility
– pH
Place a small scoop, with the spatula, of one known
white substance inside of each of these wells.

Choose one of the following:

– Aspirin
– Baking Soda Sodium Bicarbonate
– Chalk
– Contac
– Cornstarch
– Glucose
– Sudafed
– Salt
– Flour
– Tylenol
Add 3 drops of each solution to the wells. Make
sure you add the correct solution to each well.

3 drops of Benedicts Solution

3 drops of Fe(NO3)3 3.0 M
3 drops of Hydrochloric acid 3.0 M
3 drops of Iodine
3 drops of Universal Indicator Color
3 drops of Water
Place a pH strip into the well containing water
after you have tested the substances solubility.
 White Benedicts Fe(NO3)3 Hydrochloric Iodine Universal Water Methanol pH
Solution 3.0 M acid 3.0 M Indicator Solubility Solubility (use the well
Substances Color Yes, Yes, containing
somewhat, somewhat, water)
no no

Aspirin
Baking Soda Sodium
Bicarbonate

Chalk
Contac
Cornstarch
Glucose
Sudafed
Salt
Flour
Tylenol
Crimescene:

Unknown #1

Unknown #2

Unknown #3

Unknown #4

Unknown #5
 Lesson 8-9 Objectives:
Urine analysis thin layer chromatography

Evaluate evidence collected to identify what kinds

of test or equipment are needed to analyze
specific forms of evidence.

Analyze evidence, record data, and write

conclusions
 Chromatography
Basic principles
Require one static part (the stationary phase) and
one moving part (the mobile phase).

Rely on one of the following:

– Adsorption
– Partition
– Ion exchange
– Molecular exclusion
 Chromatography
Adsorption
Has a solid stationary phase and a liquid or gaseous
mobile phase.
The different solutes, carried along by the solvent, will
travel different distances through the solid.
Each solute has its own equilibrium.
– The least soluble or best adsorbed ones travel slowly.
– This results in separate bands containing different solutes.
The solvent that is put into a column is called the eluent,
and the liquid that flows out of the end of the column is
called the eluate.
Thin layer chromatography (TLC)

The stationary phase is a thin layer of a solid like silica

supported on an inert base such as glass, aluminum
foil, or insoluble plastic.

The mixture is ‘spotted’ at the bottom of the TLC plate

and allowed to dry. The plate is placed in a closed
vessel containing solvent (the mobile phase) so the
liquid level is below the spot.
 Thin layer chromatography (TLC)

TLC has advantages over paper chromatography:

– Its results are more reproducible.
– Separations are very efficient because of the much smaller particle
size of the stationary phase.

The solvent travels up the plate by capillary action.

This process is performed in a closed container to make

sure the environment directly around the experiment is
saturated with solvent vapor, and evaporation from the
plate is minimized before the experiment is complete.
 Thin layer chromatography (TLC)

The plate is removed when the solvent has reached the

top of the plate, and the position of the solvent has been
recorded before it is dried.
– These values are needed to calculate the Rf value.

The solvent travels up the plate by capillary action. The

liquid fills the spaces between the solid particles. This
technique is usually done in a closed vessel to ensure that
the atmosphere is saturated with solvent vapor and that
evaporation from the plate is minimized before the run is
complete.
Thin Layer Chromatography (TLC)

Many spots are not visible without the plates being

“developed.” This usually involves spraying the spots
with a solution that is reversibly adsorbed or reacts in
some way with the solutes.

Two examples of developing solutions are iodine in

petroleum ether and ninhydrin (useful for identifying
amino acids).
 Thin Layer Chromatography (TLC)

Iodine vapor is also used to develop plates in some cases.

Specially prepared plates can be used that fluoresce in

ultraviolet light. Once dried, the plates are placed under an
ultraviolet lamp.

Solute spots mask fluorescence on the surface of the plate

as a dark spot is observed. Some compounds have their
own fluorescence which can be used for identification.
 Lesson 10 Objectives:
Conclusions and Review
Students will work within groups to
discuss experimental results.
Students will formulate conclusions and
identify of the purpose of different
analytical tools used by Crime scene
Investigators and Forensic Technicians.
 References
Cannons J.L., Yu L.J., Jankovic D., Crotty S., Horai R., Kirby M., Anderson S.,
Cheever A. W., Sher A., Schwartzberg P.L: SAP regulates T cell-mediated help
for humoral immunity by mechanism distinct from cytokine regulation

Houck M.M., Siegel J.A.(2006): Fundamentals of Forensic Science 1 st Ed

Yin L, Al-Alem U, Liang J, Tong WM, Li C, Badiali M, Médard JJ, Sumegi J,

Wang ZQ, Romeo G. Mice deficient in the X-linked lymphoproliferative disease
gene sap exhibit increased susceptibility to murine gammaherpesvirus-68 and
hypo-gammaglobulinemia. http://www.ncbi.nlm.nih.gov/pubmed/12966553

http://www.accessexcellence.org/AE/AEPC/DNA/detection.php

https://www.amherst.edu/system/files/media/1898/2012_Manual_5c_Alu_Lab.pdf
 References
http://biotech.law.lsu.edu/map/TheFryeRule.html

http://biotech.about.com/od/pcr/a/PCRtheory.htm

http://www.carolina.com/pcr-kits/dnalc-human-mitochondrial-dna-extraction-and-
amplification-kit-with-05-ml-tubes/211236.pr

http://chemistry.about.com/od/chemistryterminology/a/What-Is-A-Control-Group.
htm

http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA460885

http://www.ehow.com/way_5255896_pcr-basics.html
 References
http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab17/lab17.html

http://www.genome.gov/10000207

http://medical-dictionary.thefreedictionary.com

http://www.nfstc.org/pdi/Subject02/pdi_s02_m02_01_b.htm

http://www.nfstc.org/pdi/Subject01/pdi_s01_m02_01.htm

http://www.niaid.nih.gov/topics/immunesystem/Pages/whatIsImmuneSystem.aspx

http://projects.nfstc.org/fse/01/01-05.html
 References
https://www.promega.com/~/media/Files/Resources/Profiles%20In%20DNA/802/
Identifying%20and%20Preventing%20DNA%20Contamination%20in%20a%20DNA
%20Typing%20Laboratory.ashx

http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html

http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html

http://www.sog.unc.edu/sites/www.sog.unc.edu/files/Supplemental
%20Materials_2012IDXX0007OD.pdf

http://www.sog.unc.edu/sites/www.sog.unc.edu/files/Supplemental
%20Materials_2012IDXX0007OD.pdf

http://www.trutv.com/library/crime/criminal_mind/forensics/dna/2.html
 References
http://webcache.googleusercontent.com/search?
q=cache:rwn16EXggG8J:media.rsc.org/Modern%2520chemical
%2520techniques/
MCT5%2520Chromatography.pdf+Royal+society+of+chemistry+chromotograph
y&cd=2&hl=en&ct=clnk&gl=us

http://www.2classnotes.com/digital_notes.asp?p=DNA_Fingerprinting