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LABORATORY
Henky
henky@unud.ac.id
Daftar Ketrampilan Klinis (SKDI 2012)
Kedokteran Forensik & Medikolegal
No Keterampilan Tingkat Keterampilan
1 Prosedur medikolegal 4A
2 Pembuatan Visum et Repertum 4A
3 Pembuatan surat keterangan medis 4A
4 Penerbitan sertifikat kematian 4A
5 Pemeriksaan selaput dara 3
6 Pemeriksaan anus 4A
7 Deskripsi luka 4A
8 Pemeriksaan derajat luka 4A
9 Pemeriksaan luar korban mati 4A
10 Pengambilan sampel vaginal swab, buccal swab, 4A
darah, urine, dan isi lambung
11 Pemeriksaan bercak darah, cairan mani, dan sperma 3
12 Fotografi forensik 3
Learning Outcomes
1. Able to collect, handle, and preserve
vaginal swab, buccal swab, blood, urine,
and gastric juice for forensic laboratory
analysis.
2. Able to explain forensic analysis on blood
fluid/stain, spermatozoa, and seminal
fluid/stain.
henky@unud.ac.id
Locard’s Exchange Principle
Locard’s Exchange Principle
“Every Contact Leaves a Trace”
“Every Contact Leaves a Trace”
henky@unud.ac.id
Biological Evidence
henky@unud.ac.id
Forensic Analysis of Blood
1. Visual examination of evidence
2. Presumptive screening test (Is it blood?)
3. Confirmation test (Seriously, is it blood?)
4. Determine species origin (human blood?)
5. Identify the blood (whose blood is it?)
henky@unud.ac.id
Blood Analysis Steps
Presumptive Stain /
Catalytic Color Tests
Fluid?
+ - Blood (-)
- + Blood (+)
- + Blood Group
Chemical Analysis
Presumptive test (screening)
Probably blood
Confirmatory test
Definitely blood
henky@unud.ac.id
Presumptive Tests
Benzidine (Adler Test)
Phenolphthalein (Kastle-Meyer Test)
Tetramethylbenzidine and Hemastix®
Fluorescence Test
Chemiluminescence Test
+ On henky@unud.ac.id
Benzidine Test
Blood + H2O2 H2O + On
+ On
henky@unud.ac.id
Fluorescence Test
For cleaned up or washed spatter pattern
Fluorescin + Oxidant + Blood Fluorescein
Fluorescein will fluoresce when treated with UV
or Alternative Light Source (ALS) 425 – 485 nm
+ On
henky@unud.ac.id
Chemiluminescence Test
For cleaned up or washed spatter pattern
Luminol + Oxidant + Blood light is produced
(+) : blue-white to yellow-green in darkness
+ On
I VE
SIT
E N
T S
M OS
E
TH
henky@unud.ac.id
Confirmatory Tests (1)
Targeting the non-protein heme
group of Hb Porphyrins
Heme Hexavalent Iron Atom
Binding with 6 different entities:
4 Nitrogen atoms (Ring)
1 Histidin
1 H2O or O2
These last two positions are used
in the formation of crystals
henky@unud.ac.id
Confirmatory Tests (2)
1. Takayama test
2. Teichmann test
3. Wagenaar test
+ On
henky@unud.ac.id
Takayama Test
• Heme is gently heated with pyridine under
alkaline conditions in the presence of a reducing
sugar (saturated glucose) Crystals of pyridine
hemochromogen (yellowish pink feathers)
• (Figure 8.6A) are formed
+ On
henky@unud.ac.id
Teichmann Test
• Heating dried blood in the presence of glacial
acetic acid and a halide (usually chloride) to
form the hematin derivative Hemin Crystals
(rhombic in shape and brownish in color)
+ On
henky@unud.ac.id
Wagenaar Test
• Heating dried blood in the presence of acetone
and a halide (usually chloride) to form the
hematin derivative Hemin-acetone crystals
(rhombic in shape and brownish in color)
+ On
henky@unud.ac.id
Forensic Analysis of Blood
1. Visual examination of evidence
2. Presumptive screening test (Is it blood?)
3. Confirmation test (Seriously, is it blood?)
4. Determine species origin (human blood?)
5. Identify the blood (whose blood is it?)
SerologyTest
Serology Test
henky@unud.ac.id
Serology (1)
Most methods test for Serum Proteins
Serum proteins are found in all animals,
but are slightly different
Species ID methods based on antigen-
antibody interactions
Species Origin Determination
henky@unud.ac.id
Serology (2)
henky@unud.ac.id
Methods
1. Ring Precipitin Test (Uhlenhut)
2. Ouchterlony Double Diffusion
3. Crossed-Over Electrophoresis
henky@unud.ac.id
Ring Precipitin Test (1)
Making Human Antiglobulin Serum
henky@unud.ac.id
Ring Precipitin Test (2)
Antiserum in bottom
layer
henky@unud.ac.id
Ring Precipitin Test (2)
henky@unud.ac.id
Ouchterlony Double Diffusion
Human blood
Human
antiserum
Not human
blood
Antiserum placed in center
Several bloodstains tested at one time
White line means antiserum and blood match
henky@unud.ac.id
Crossed-Over Electrophoresis
Gel
Holes
henky@unud.ac.id
Crossed-Over Electrophoresis
henky@unud.ac.id
Identify The Blood
If a stain is blood, and it is human blood,
then whose is it?
1. Blood Group Markers
2. Protein / Enzyme Markers
3. DNA
henky@unud.ac.id
Blood Group Markers
• Erythrocyte surface antigen
– ABO, MNS, Rhesus, Duffy, Kell, Kidd, etc
• Protein serum system
– Gm, Gc, Haptoprotein, etc
• Erythrocyte enzyme system
– PGM, AK, ADA, PCE, EAP, GPT, 6-GPD, etc
• Leucocytes antigen
– HLA
• Others
– Secretor/non secretor, platelets antigen
henky@unud.ac.id
ABO Markers (1)
Antigens (agglutinogens) on RBC are glycoproteins and
are attached on the surface of red blood cell
Antibodies (agglutinins) are present in the plasma
henky@unud.ac.id
ABO Markers (2)
henky@unud.ac.id
ABO Markers (3)
Testing is similar to species test
An anti-A, anti-B, or anti-AB antiserum
(containing antibodies) is reacted with the
blood stain to detect blood cells
A, B, or AB blood cells are reacted with a
blood stain to detect antibodies
Tests can get complicated with absorbing and
releasing cells
Final step is usually testing for agglutination
henky@unud.ac.id
Direct Test
henky@unud.ac.id
Indirect Test
(Absorption Elution)
henky@unud.ac.id
henky@unud.ac.id
Protein or Enzyme Markers (1)
Some protein or enzymes can be in different
forms (different shapes)
These differences can be detected by
separating the protein in a gel by
electrophoresis
henky@unud.ac.id
Protein or Enzyme Markers (2)
+ Ladder Type 1 Type 2 Type 3
henky@unud.ac.id
Another Uses of Forensic Serology
Paternity Cases:
• The babies get accidently exchanged
• Kidnapped baby
• Man suspected as biologic father
• Husband confuse about the baby
• Disputed paternity
– Mendel Law
– Paternity exclusion
henky@unud.ac.id
ABO Mendelian Inheritance
henky@unud.ac.id
Case (1)
Phenotypes
Phenotypes
Father Mother
A AB
Child
henky@unud.ac.id
Case (2)
Phenotypes
Phenotypes
Father Mother
O AB
AB
Child
henky@unud.ac.id
Percentage of Blood Types
Antigen
Blood Type Antibody % Population
(Blood Group)
A A Anti-B 40
B B Anti-A 10
AB A&B None 5
O H Anti-A & 45
Anti-B
henky@unud.ac.id
Blood Group Markers Probability (%)
RBC Antigen
MNS 32.1
Rhesus 28.0
Kidd 19.0
Duffy 18.0
ABO 17.6
Kell 3.3
Lutheran 3.3
Protein Serum
Gc 24.7
Hp 17.5
Glm 6.5
Km 6.0
RBC Enzyme
PGM 25.3
EAP 21.0
GPT 19.0
Glyoxalase 18.4
Esterase D 9.0
AK 4.5
ADA 4.5
HLA 94.0
henky@unud.ac.id
Rape
• Serological evidence is crucial
• Forceful physical contact
Transfer of evidence:
– Blood, Semen, Perspiration, Urine
– Hair and Nail
• Proving sexual intercourse by finding
semen
henky@unud.ac.id
Semen
Semifluid mixture of cells, amino acids, sugars,
salts, ions, and other organic and inorganic
materials
Elaborated as a heterogeneous gelatinous mass
Contributed by the seminal vesicles, the prostate
gland, and Cowper’s glands
Ejaculate volumes of human males range from 2
to 6 milliliters and typically contain between 100
and 150 million sperm cells per milliliter
henky@unud.ac.id
Semen Examination
1. Sperm Cells
Microscopic examination:
• Direct Technique
• Indirect Technique Staining
2. Seminal Fluid Protein Rich Bodily Fluid
Chemical analysis:
• Crystal Tests: Spermine & Choline
• Enzym Markers: SAPs
• Zinc Test
Electrophoresis / Immunological methods: PSA
henky@unud.ac.id
Semen Stains
Visual: yellowish-white
Tactile: stiff
Odor: ~ chlorine
ALS: fluorescence
henky@unud.ac.id
+ On
Semen Analysis Steps
Presumptive Stain /
Chemical Analysis
Fluid?
+ - Semen (-)
- + Semen (+)
Blood Group
Presumptive Tests
Crystal Tests
Florence
Berberio
Puranen
Enzym Markers
Seminal Acid Phosphatase
Zinc Test
henky@unud.ac.id
Florence Test
Choline + lugol (KI) Crystals of choline
periodide (brownish needle rhomboid shaped)
henky@unud.ac.id
henky@unud.ac.id
Berberio Test
Spermine + saturated picric acid Crystals of
spermine picrate (yellowish neddle shaped)
henky@unud.ac.id
henky@unud.ac.id
Puranen Test
Spermine + Naphtol S Yellow Crystals of
spermine flavinate (yellowish neddle shaped)
henky@unud.ac.id
Acid Phosphatase Test
Acid Phosphatase will hydrolyse Alpha-Naphthyl
Phosphate (Substrate) to Alpha-Naphthol
Alpha-Naphtol + Brentamine Fast Blue (Chromogen /
Color Developer) Azo (dark purple) within 30 seconds
+ On
henky@unud.ac.id
henky@unud.ac.id
Zinc Test
Zinc + Tris (hydroxymethyl) aminomethane +
PAN (1-(2-Pyridylazo)-2-naphthol) Pink
+ On
henky@unud.ac.id
+ On
Confirmatory Tests
Microscopic Examination
Direct Technique Sperm Cells (Motile)
Christmas Tree / Malachite Green Stain Sperm
Cells (Immotile)
Baecchi Stain Sperm Cells on clothing, bedding, etc
Electrophoresis
PSA / p30
Immunological methods
PSA / p30
henky@unud.ac.id
Direct Technique
henky@unud.ac.id
henky@unud.ac.id
Christmas Tree
Distinguishing sperm cells
from extraneous material such
as epithelial cells
Two dyes used:
Green : Malachite Green/
Picroindigocarmine (PIC)
Stains tails
Red : Eosin / Nuclear Fast Red
(NF)
Stains heads
henky@unud.ac.id
henky@unud.ac.id
Baecchi
Sperm cells on clothing, bedding, etc
Two dyes used:
Acid Fuchsin
Methylene Blue
+ On
henky@unud.ac.id
henky@unud.ac.id
Prostate-Specific Antigen
PSA is a protein produced by the prostate gland
Confirming semen in samples that do not contain
sperm cells
Test relies on antigen (PSA) and antibody
interaction
henky@unud.ac.id
PSA Test by Electrophoresis
henky@unud.ac.id
PSA Rapid Test Device
Membrane Based Immunochromatography
henky@unud.ac.id
henky@unud.ac.id
Results
Invalid Results
Research
Year Researcher Rapid Test Sen Spe PPV NPV Time
2004 Khaldi, et al PSA Check-1 99.4 98 99.4 98 48 h
2007 Peonim, et al One-Step PSA 85 85 89 79 -
2009 Hobbs, et al ABA-Card 100 96 - - -
2009 Laux, et al Seratec - - - - 72–96 h
2013 Peonim, et al InTec Products 80.4 92.3 77.6 93.5 -
henky@unud.ac.id
If Semen, Whom Does it Belong to?
• Blood Group Type
– Secretor : ABO, Lewis Blood Typing
– Methods: Absorption Inhibition
• DNA : Y-STR
henky@unud.ac.id
Genetic Markers in Blood / Semen
ABO blood typing and protein analysis
may help eliminate a suspect (exclusion)
Since there are only a small number of
types (ABO = 4 types), a match does not
mean the stain definitely came from a
certain person
DNA testing is very specific, thus it can
identify a person, and is becoming just as
easy as the above tests
henky@unud.ac.id
References
1. Greenfield A, Sloan MM, Spaulding RP. Identification of Blood and
Body Fluids. In: James SH, Nordby JJ, Bell S, editors. Forensic
Science An Introduction to Scientific and Investigative Techniques.
Fourth Edition. Boca Raton: CRC Press Taylor & Francis Group;
2014. p. 206-28.
2. Yudianto A. Panduan Praktis Serologi Forensik. Surabaya: Global
Persada Press; 2013.
3. Sudiono S. Kumpulan Makalah dan Penelitian Ilmiah Ilmu
Kedokteran Forensik. Jakarta: Departemen Ilmu Kedokteran
Forensik dan Medikolegal FKUI-RSCM; 2008.
4. Gaensslen RE, Camp FR. Identification of body fluids. In:
Sourcebook in forensic serology, immunology, and biochemistry.
U.S. Department of Justice: National Institute of Justice; 1983.
henky@unud.ac.id
henky@unud.ac.id
THANK YOU