Techniques of Identification (Part 2) Forensic Serology

(online class, 2020)

 Involves the examination and analysis of body fluids
Odontology
Body fluids analysed
 Blood
 Use of dentition in the identification of individuals  Saliva
 Allowed for the comparison of dentition with pattern injuries  Vomitus
known as bite mark analysis
 Feces
 Was 1st used as a means of identification in 1st century A.D.
 Urine
 Gained widespread acceptance as a means of identification in the
 Semen
19th century
 Vaginal Secretions
 In 1849, dental identification was used in the U.S. For
identification purposes
Identification of Blood Stains
 In 1849, dentition was 1st used in a mass casualty incident (Vienna
 Careful Visual Examination
Opera House Fire)
o Determine the material and make of the stained material
o Note the color of the stain and the surface
Importance o Note the directionality of the stain
 The number of possible combinations possible with 32 teeth and 5
surfaces make dental records virtually unique  Directionality
 Enamel is the hardest substance of the human body and may o Tells the direction of the blow as well as the velocity of the
outlast all other tissues. wounding instrument

Dental Records  Application of a presumptive screening test

 PD 1575 requires dentist to keep dental records of their patients o Presumptive tests for blood
 If 10 years has passed since the last entry, the dentist is mandated  Catalytic color test
to submit their records to the NBI  Depends of the peroxidase-like activity of the
heme group of haemoglobin
Causes of unreliability:  Includes the following:
 Dentist may keep incomplete records  Benzidine Test (Adler Test)
 No uniformity of nomenclature  No longer in use because it is
 No agency is tasked for enforcement carcinogenic or cancer causing to the
 Lapse of time due to poor dental follow up would make records examiner
unreliable.  Phenophthalein Test ( Kastle Mayer) (specific)
 1:100,000 sensitivity
Other techniques  O-Tolidine
Gas Chromatography  Tetramethylbenzidine (most sensitive)
 Technique of separating a mixture of compounds using a solvent  Similar to Benzidine test but modified to
(liquid phase) and a chromatographic packing material (stationary prevent carcinogenicity
phase)  Leucomalachite Green (specific)
 Usually paired with Mass Spectrometry  1:300,000 sensitivity
 Chemiluminescence
High Performance Liquid Chromatography  Process by which light is emitted as a
product of chemical reaction
 Technique of separating a mixture of volatile compounds after
being flash evaporated  Example is Luminol
 Has sensitivity of 1: 5,000,000
 Usually paired with Mass Spectrometry
 Limitation is it has a cross reaction with
bleach
Mass Spectrometer
 Fluorescence
 Measures mass to charge ratio of ions
 When a chemical substance is exposed
 Results are given as a mass spectrum to a certain wavelength of light and light
is emitted as a by-product.
Atomic Absorption Spectrophotometry  Excitation wavelength is 520 nm
 Technique that measures concentration of elements.  Emission wavelength is 480 nm
 Very sensitive and can read as little as 1 part per billion  Example is Fluorescein
 Uses of wavelengths of light that is absorbed by a particular
element.  Advantages of Chemiluminescence/Fluorescence
o When blood has been cleaned or not clearly visible, both
Dog Scent Identification techniques can still show the blood pattern.
o When the purpose is to show the extent of blood for
purposes of documentation
 Application of a confirmatory test  HbF (Fetal haemoglobin)
o Crystal Test (formation of haemochromagen crystals)  Useful in the determination if the victim is very
 Teichmann Test young such as cases of infanticide.
 Takayama Test  HbS (haemoglobin found in persons with sickle cell
o Microscopic Analysis anemia)
 Rhombic or diamond shaped crystals are seen on  Usually seen in African Americans
microscopy after application of the crystal test
 Haptoglobin
 Determine the biologic or species origin o Alpha 2 globulin class
o Species Origin Determination o Not so useful anymore due to the advent of DNA technology
 Serum Protein Analysis
 The following may be used for testing:  Enzyme Markers
 Albumin o Uses flow cytometry
 Alpha -1 globulins o Rarely utilized nowadays due to the easy access of DNA
 Alpha -2 globulins analysis
 Beta globulins
 Gamma globulins Semen
 Most useful  Semifluid mixture of sperm cells, amino acids, sugar, salts, and
 Also known as antibodies organic compounds
 The following techniques may be utilized  Ejaculate ranges from 2-6 ml with an average of 3.5 ml/ejaculation
 Ring Precipitin Tests  100-150 million sperm cells / ml.
 Ouchterlony Double Diffusion Tests  According to Solis: 60 million/ml
 Crossed over electrophoresis
 Nonserum Protein analysis Sperm Cell
 Antihuman hemoglobin antibody  Approximately 55 um
 In this test antibodies that target  Ovoid in shape
human blood are mixed with
human blood. A positive result Till When Can it be Detected?
would cause the red blood cells to
 Intact Sperm
burst.
 up to 26 hours after intercourse in the vagina
 Characterization of blood using genetic markers
 Up to 5 hours in oral or rectal vaults
o Genetic Markers in Blood o Sperm Heads
 Antigen Based Markers  Up to 7 days in vaginal cavity
 ABO system  2-3 days in the anus and rectum
 Most important  24 hours in the oral cavity
 Discovered by Land-steiner in 1900
 Most useful as a tool for exclusion Acid Phosphatase
 Seen in other body fluids if the subject is a  Used for the presumptive identification of semen
secretor  Uniquely high levels in seminal fluid
 Detected in the vagina up to 72 hours post coitus
 Lewis system  Reagent used: Brentamine Fast Blue test
o 2 antigens – Lewis a/Lewis b
o Provides insight to secretor status or whether other bodily Other Presumptive Tests for Semen
fluids have ABO antigens present or not  Florence Test – Depends on action of iodine on choline
o Le(a-b+) – ABH secretors – 72% of population
 Berberio’s Test – formation of yellow crystals using picric acid
o Le(a+b-) – ABH non secretor – 22%
o Le(a-b-) – 6 % of popu.  Puramen Reaction – Yellow Crystals form in the presence of
Puramen’s reagent
 80% secretor
 20% nonsecretor
Confirmatory Tests
o Le(a+b+) – very rare.
 Prostate Specific Antigen (PSA)
 Rhesus system  Gamma-seminoprotein
o Wiener (Rh)  Secreted by the cells lining the prostate glands
 This is the positive or negative in your blood type. This o Visualization of Spermatozoa
is not very useful as it does not follow Mendellian  Sperm Cell Identification
Genetic rules like ABO.  Dye (Christmas tree stain) is applied to the slide
o Fisher – Race (C, c, D, E, e) o Sperm cell tail – yellow green
o Midpiece – blue
 Protein Markers o Acrosome – pink
o Hemoglobin o Head – dark red / purple
 HbA (Adult haemoglobin)
 Other Dye  Biological sample - any organic material originating from a
o H & E Stain person's body, even if found in inanimate objects, that is
o Hankin’s Method (Carbol fuchsin) susceptible to DNA testing.
o Ganguli’s Method (Erythrocin and Malachite green) o Includes :
 Blood
Test for whether the sample is of human origin or not  Saliva and other body fluids
 Biological Test of Farnum or Precipitin Test  Tissues
 Hairs and bones
Importance of Semen Analysis o DNA profile - genetic information derived from DNA testing
 Useful in crimes involving sexual assault like of a biological sample obtained from a person, which
 Rape biological sample is clearly identifiable as originating from
 Anti VAWC that person
o DNA evidence - the totality of the DNA profiles, results and
 Anti Trafficking Cases
other genetic information directly generated from DNA
 Stalking testing of biological samples
 DNA as EVIDENCE
Other Body Fluids  Allowed expressly by the Rule on DNA Evidence
o DNA testing-verified and credible scientific methods
Saliva  Include
 Slightly alkaline secretion  Extraction of DNA from biological samples,
 1 to 1.5 liters of saliva is produced per day  The generation of DNA profiles
 Amylase (an enzyme used to digest starch) is used to test for it  Comparison of the information obtained from the
 Starch-Iodine Test DNA testing of biological samples for the purpose
 Phadibas reagent of determining, with reasonable certainty,
o Limitation: whether or not the DNA obtained from two or
 Amylase is not only present in saliva more distinct biological samples originates from
the same person (direct identification) or if the
 It is also found in secretions of the pancreas
biological samples originate from related persons
(kinship analysis)
Urine
 Presumptive test uses alternative light sources DNA Testing
 The test relies on urea and creatinine  Discovered in 1984 in University of Leicester by Sir Alec Jeffries
Feces
 Used first to solve the Enderby Murders in England
 Depends on repetitive sequences which are repeated over the
 Characteristic smell is caused by Skatole
chromosome and are inherited and conserved from familial lines
 Urobilinogen – most common means utilized for identification
 Occurs either in tandem or dispersed
 Limitation:
 Comprise 50% of the human genome
 Infant < 6 months
 Example of sequences
 Carnivorous/omnivorous people only
 Pseudogenes
Vomitus  Alu repeats
 No specific test for presence of vomitus  Duplications of large stretches of DNA
 Analysis of stomach contents  Interspersed repeats occurring in the centromere
 Simple sequence repeats
Vaginal Secretions
 Important in cases of rape where allegations of foreign body Simple Sequence Repeats
insertion are made  Currently the gold standard in DNA fingerprinting
 Tests for glycogenated epithelial cells or the cells lining the vaginal  Utilizes the high variability of the repeating units as basis for
canal comparison
 Uses PAS (Periodic Acid Schiff) reagent  People have differing numbers of allelic repeat units inherited
 Limitation: from their parents.
 Glycogen depends on the stage of the menstrual cycle  Could either be:
 Microsatellites
DNA o Short tandem repeats (STR)
 The genetic material of the body which serves as its blueprint
o 2-7 bp in each tandem repeat unit
 DNA stands for deoxyribonucleic acid
 Minisatellites
o Variable number of tandem repeats (VNTR)
 The chain of molecules found in every nucleated cell of the body.
o 9-80 bp in each tandem repeat unit
 The totality of an individual's DNA is unique for the individual,
except identical twins.
o Definition of Terms (From rules on DNA Evidence)
DNA STR Analysis
 Uses multiple loci or areas in the DNA strand
 Limited number of these variations/loci
 13 core microsatellite loci – basis for DNA fingerprinting (US)
 10 core microsatellite loci – basis for DNA fingerprinting (UK)
Steps of DNA Analysis
 Uses Polymerase Chain Reaction (PCR) to mass duplicate a sample
DNA
 Results to formation of amplicons which are copies of the original
sample
o Electrophoresis
 Technique of separating the sample depending on sample size.
 Capillary Electrophoresis – modern equivalent which uses smaller
sample sizes
o Short Tandem Repeat Analysis
Confidentiality of Results
 DNA profiles and all results or other information obtained from
DNA testing shall be confidential
 only be released to any of the following, under such terms and
conditions as may be set forth by the court:
o Person from whom the sample was taken;
o Lawyers representing parties in the case or action where the
DNA evidence is offered and presented or sought to be
offered and presented
o Lawyers of private complainants in a criminal action
o Duly authorized law enforcement agencies
o Other persons as determined by the court

Medicolegal Applications
 Identification
 Exclusion of Suspects
 Paternity Testing

Paternity Testing
 DNA results that exclude the putative parent from paternity shall
be conclusive proof of non-paternity.
 If the value of the Probability of Paternity is less than 99.9%, the
results of the DNA testing shall be considered as corroborative
evidence.
 If the value of the Probability of Paternity is 99.9% or higher, there
shall be a disputable presumption of paternity

Rules in DNA Evidence

Application for DNA Testing Order
 Motu proprio
 On application of any person who has a legal interest in the
matter in litigation
 Requisites
o A biological sample exists that is relevant to the case
o The biological sample:
(i) was not previously subjected to the type of DNA testing
now requested

(ii) was previously subjected to DNA testing, but the

results may require confirmation for good reasons

 The DNA testing uses a scientifically valid technique

 The DNA testing has the scientific potential to produce new
information that is relevant to the proper resolution of the case
 The existence of other factors, if any, which the court may
consider as potentially affecting the accuracy or integrity of the
DNA testing.

Probative Value of DNA Evidence in Paternity Testing

 DNA results that exclude the putative parent from paternity shall
be conclusive proof of non-paternity.
 If the value of the Probability of Paternity is less than 99.9%, the
results of the DNA testing shall be considered as corroborative
evidence.
 If the value of the Probability of Paternity is 99.9% or higher, there
shall be a disputable presumption of paternity