FORENSIC BIOCHEMISTRY

• Forensic Biochemistry  branch of Biochemistry that deals

with law (Civil or criminal law)

• Forensic investigations  solve disputes in criminal acts. eg.

drug abuse, suicide, paternity dispute etc

• Sample could be physical or biological tissue to determine

the facts about what happened, when it happened where it
happened, and who was involved

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 Applications of forensic science
• Identification of suspects via DNA finger
prints at the crime site

• Exoneration of wrongly accused people

• Establishing timing and circumstances of

a crime

• Settling of paternity disputes

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 Classification of forensic evaluation
1. Post-mortem analysis: evaluates chemicals, drugs, volatile
substances like CO and their metabolites, that contributory factors in
the cause of death

2. Systematic toxicological analysis (STA): Determines the role of

chemicals/drugs in blood or other specimens, in evaluating human
performance or behaviour.

3. Forensic urine drug testing: Detects the presence of drugs and their
metabolites in the urine to demonstrate their prior use or abuse.

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 Analytical methods in forensic evaluation

2 most common strategies:

1. Screening tests: to detect several analytes/metabolites

2. Confirmatory tests:
The analyte is detected and quantified

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 Samples used in forensic science

• Choice of sample depends on:

1. Analyte being sought
2. purpose and ease of the analysis
3. Type of information required

NB: For post-mortem analysis, sample should be collected before

embalmment because the process may destroy/dilute poisons,
resulting in poor detection.

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 Samples used in forensic science
1. Urine - mostly used in the screening tests.

2. Oral fluid and hair - because of their ease of collection

3. Blood: mostly used in confirmatory tests – blood stain, type of stain,

shoe prints, etc. reveal a lot about the crime in question.

4. Tissue biopsy/autopsy, e.g. liver, brain, kidney, heart, bile, gastric

juice, etc.

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Advantages & disadvantages of samples used in forensic science
Samples Advantages Disadvantages
Blood • Large database for correlation with analyte
effect • Some substances are rapidly
• Semi-invasive cleared
• Several collection sites
• Several methods for analysis
Urine • High conc. of drugs & metabolites • 2-3 days detection period
• Several testing methods • No dose-conc. relationship
• Results are often reliable • Liable to contamination
Oral fluid • Easily accessible • Liable to contamination esp
• detection of recent drug use if patient is on other drugs
• Result related to behaviour/performance • Detection window may be
• Detects drugs & metabolites shorter
Liver • Many drugs & their metabolites are present • Invasive procedure is
at high conc required to take sample

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Samples Advantages disadvantages
Brain • Target organ for several drugs • Poor correlation with drug effect
• Extensive sample preparation
• Unequal drug distribution

Vitreous • Easily analysed • Not easily accessible

humour • Many drugs can be measured • Difficult to correlate drug concentration
with blood concentration

Hair • Gives long term history of drug use • Difficult to differentiate exterior and
• Easy to collect interior drug concentration
• Analytical & interpretive difficulties

Exhaled • Easy to collect • Only used for antemortem analysis

air • large volume always available for • Good for only volatile substances-
both qualitative & quantitative ethanol, CO, many xenobiotics
analyses

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 Substances commonly detected in forensic analysis
 Drugs likely to be abused e.g. ethanol, opioids, CNS stimulants,
hallucinogens, cannabinoids, hypnotics, anxiolytics,
antidepressants, etc.

 Environmental toxins e.g. CO, pesticides, cyanides, etc,

which are detected using systematic toxicological analysis
(STA)

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 Criteria for selecting the type of method for forensic analyses

1. Sensitivity of the test - to detect and quantify the analyte.

2. Specificity of the test - to differentiate the analyte from several others

in the same sample

3. Competency of the analyst: in terms of the knowledge, skills and

experience

4. Reliability of the method - to be easily reproduced all the time

5. Others – e.g. safety, cost and ease of performance

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 METHODS USED IN FORENSICS
• Enzymatic assays e.g. acid phosphatase, alcohol oxidase tests
• Xenobiotic analysis – biological monitoring to detect the
xenobiotics/their metabolites in various samples
• Determination of metals (Cu, Pb, Zn, Cr) or non-metals (fluoride) – by
AAS, and potentiometer
• Immunoassays, such as ELISA, RIA, IHC & FIA
• Chromatographic techniques – eg. TLC, GC, HPLC, GC-MS
• Molecular techniques – eg. DNA finger print, DNA analysis
• Metabolomics – detection and quantification of cellular metabolites
• Biosensors – eg. Use of calorimetry to detect the presence specific
chemicals 11
 Enzymatic methods
• Alcohol oxidase test, detects the presence of alcohol (ethanol) in
saliva/blood at a concentration ≥ 0.02% using test strip method

• Acid phosphatase test, detects the presence of semen in cases of

gender-based violence such as rape, forced marriages, abortions, etc.

NB: Concentration of AP in semen is 1000X greater than in any

other body fluid
 Xenobiotics Analaysis
 Metabolites of xenobiotics are determined in blood, urine, breath (for volatile
substances), hair, nails (for trace elements e.g. selenium), milk (in lactating
women), adipose tissue, etc.

 Determination of exposure to xenobiotics is called biological monitoring

• This is useful in determining the effectiveness of preventive measures such as
personal hygiene, protective clothing, etc.
• When samples used to assess xenobiotics are obtained from a work place  work
place monitoring

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 Indicators of exposure to Xenobiotics
• The 2 major considerations are the type of sample and type of analyte

• Entry site determines the sample type e.g. exposure to asbestos in the air by inhalation
manifests as lung lesions
- Xenobiotic may not be present in the analyte e.g. exposure to aniline through the skin 
raised methaemoglobin (this is called an adduct)

• Adducts are formed when analyte is different from the parent compound e.g.
carboxyhaemoglobin from CO exposure, methaemoglobin from aniline and
nitrobenzene exposure, DNA methylation during carcinogenesis etc

• Xenobiotics are metabolized by phase I and II reactions  water soluble metabolite

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 Determination of metals

• Metals such as chromium, copper, lead, lithium, zinc, in the

blood/urine, are determined by atomic absorption
spectrometry after the process of wet ashing

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 Immunoassays

• Immunoassays (ELISA, RIA, IHC & FIA) are reliable

diagnostic tools in forensic studies

• It involves the use of antibodies to detect and quantify

xenobiotics and/or their metabolites in the sample, using a
specific antigen-antibody interactions.
Examples of drugs analyzed via immunoassays

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 Chromatographic Techniques
 Thin layer chromatography (TLC) identify and compare compounds present
in different samples.

 Gas Chromatography (GC) analyzes volatile compounds based on their

chemical properties. It has high resolution speed compared to TLC

 HPLC partition the compounds in stationary and mobile phases. It has high
resolution speed and conveniency compared to GC.

 GC-MS – The gold standard of analytical methods in forensic tests. It

determines and quantify the “unknown” in any sample with high precision. It
is widely used in pathologic forensic, crime scene testing, drug of abuse
screening, etc.
 Molecular Techniques
Examples:

1. DNA finger printing/DNA profiling

2. DNA analysis from bone, skin follicle, hair strand, blood, etc

NB: DNA is the building block of an individual’s entire genetic makeup. It has
unique sequence of base-pairs (microsatellites) that differs (at least by 1%) in
every two individuals, except in the identical twins.
 DNA fingerprinting/DNA profiling
• Is a way of identifying microsatellites that differ in the DNA of every
individual.

• DNA fingerprinting has high success rate and very low false-positive rate.

• DNA fingerprint is derived by three modalities:

• Short tandem repeat (STR),
• Single nucleotide polymorphism (SNP)
• Restriction Fragment Length Polymorphism (RFLP)
 Short Tandem Repeat (STR)
This is a sequence of DNA that consists of adjacent repeats of a set of nucleotides.

• Polymorphism occurs when homologous STR loci differ in the number of repeats
between two different individuals.

• By identifying repeats of a particular sequence at specific locations in the genome, it is

possible to create a genetic profile of an individual.
Steps of Short Tandem Repeat Analysis

- Obtain the biological specimen

- Extract the DNA
- Target specific STR on the chromosome
(Primer Design)
- Run the polymerase chain reaction (PCR)
- Visualize the product of PCR
 Single Nuclear Polymorphism (SNP)
 SNPs are genetic markers of DNA profiling responsible for ~ 85% of
human genomic variation.

They are much shorter DNA sequences (50bps) and more abundant
compared to STRs. There are four main classes of SNPs: Identity-testing
SNPs, Lineage-informative SNPs, Ancestry-informative SNPs, &
Phenotype-informative SNPs
 Finger print determination
• Physical evidence to be tested may be any of the following
• Newspaper, Glass bottle, Eyeglasses
• Disc, Glossy magazine
• Surfaces like bite mark, finger nails, hat, mask, table, wall etc
• The detection techniques are
1. Iodine Fuming: good for paper evidences, iodine fixes print,
but the method is not popular currently
2. Cyanoacrylate (Superglue) Fuming: vapor of the glue is used
to get the latent print
3. Powders: dusting powder is used to enhance ridge details of
latent print
4. Ninhydrin Spraying: the most common spray method used to
reveal latent prints
5. Detection machines in the form scanners etc
 Steps in DNA fingerprinting
1. Isolation of DNA: extraction from cell
2. Cutting, sizing and sorting: usually enzymes
are used-restriction enzyme whose action is on
specific letter sequence
3. Separating DNA fragments using gel
electrophoresis
4. Transfer of DNA to nylon (nitrocellulose
paper)-this is southern blotting
5. Probing: adding probe which is a small
sequence of radioactive labelled DNA
segment capable of sticking to DNA fragments
on the nylon
6. Fingerprinting: the probe used is of 33 letter
sequence, after adding the probes, the gel is
placed against an x-ray film in the dark and
left overnight. The x-ray film records
radioactivity of where the probe stick with
 Application of DNA fingerprinting
1. Diagnosis of inherited diseases and developing their treatment
2. For personal identification
3. Solving paternity disputes, crime cases, etc
The advantages of fingerprinting are that it is accurate and cannot be altered.
However, it has some limitations/challenges including:
4. Sample is easily contaminated
5. Procedure is very complex
6. In animals like dogs is difficult to use fingerprinting to establish a carrier of
disease causing allele
7. There is need for an expert person
8. Time consuming procedure
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Steps in DNA Analysis
 Metabolomics
 Metabolomics is the study of the entire collection of metabolites of a cell
based on their spectral peaks and calibration curves. .

 Metabolite can be a peptide, oligonucleotide, lipid, alcohol, ketone, sugar,

amine, alkaloid, steroid, drugs or xenobiotic.

 Metabolome plays a role in toxicology testing, drug compliance, genetic

disorder tests, drug phenotyping, and blood analysis.
 Biosensors
 A biosensor is a device that detects the presence of
specific chemicals in an area via calorimetry,
electrochemistry, etc.

 Biosensors are used to identify genetic disorders in

forensic sciences, which helps to find the accused out of
random suspects.
Biochemical method used in the estimation of time of death
• The common biochemical marker is potassium level in the vitreous humour.

However, it has some limitations such as:

• Potassium concentration may differ between the left and right eye
• Presence of electrolyte disturbance ante-mortem, but this can be eliminated
by excluding cases with a vitreous humour urea level of 100mg/dl. Because
high vitreous urea reflects ante-mortem retention not due to post-mortem
changes

• If ante-mortem electrolyte imbalance is excluded, there is linear relationship

between K concentration in vitreous humour and the time after death of up
to 120 hours
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Unravelling the circumstances of death and possible poisoning agent
Pathological observation Possible poisoning agent (s)
Burns around mouth, lips, nose Acids or bases (lye, potash, hydroxides)
Darkness of the skin around face and neck Aniline, nitrobenzene
Severe, unexplained diarrhoea Metals (arsenic, mercury, copper, etc.)
Dilated eye pupils Atropine (Belladonna), Scopolamine
Odour of disinfectant Carbolic acid or other phenol
Bright cherry red skin Carbon monoxide
Quick death, red skin, with peach odor Cyanide
Vomiting, abdominal pain Food poisoning
Diarrhoea, vomiting, abdominal pain Metallic compounds
Convulsion Nicotine, Sodium fluoride
Odour of garlic Oxalic acid, phosphorous
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THANK YOU

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