FORENSIC

LABORATORY

Henky

Department of Forensic Medicine

Faculty of Medicine Udayana University

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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.

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 Locard’s Exchange Principle
Locard’s Exchange Principle
“Every Contact Leaves a Trace”
“Every Contact Leaves a Trace”
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Biological Evidence

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 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?)

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 Blood Analysis Steps
Presumptive Stain /
Catalytic Color Tests
Fluid?

+ - Blood (-)

Confirmatory Microcrystal Tests

- + Blood (+)

Human Precipitation Test

- + Blood Group
 Chemical Analysis
 Presumptive test (screening)
 Probably blood
 Confirmatory test
 Definitely blood

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 Presumptive Tests
 Benzidine (Adler Test)
 Phenolphthalein (Kastle-Meyer Test)
 Tetramethylbenzidine and Hemastix®
 Fluorescence Test
 Chemiluminescence Test

 + On [email protected]
 Benzidine Test
 Blood + H2O2  H2O + On

 Oxidized Benzidine  Dark Blue

 Interpretation:
 Positive result  probably blood
 Negative result  definitely
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not blood
 Phenolphthalein Test
 Blood + H2O2  H2O + On

 Oxidized Phenolphthalein  Pink

 Interpretation:
 Positive result  probably blood
 Negative result  definitely
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not blood
 Tetramethylbenzidine
 3,3′,5,5′-tetramethyl derivative of benzidine in an
acid medium (Acetic Acid)
 Stain + H2O + Hemastix®
 Positive result  blue - green
 Negative result  yellow

 + On
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 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

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 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

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 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

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 Confirmatory Tests (2)
1. Takayama test
2. Teichmann test
3. Wagenaar test

 + On

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 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

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 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

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 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

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 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
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 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

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 Serology (2)

 Antigen = Serum Protein in

Blood Sample
 Antibody = Produced when
foreign serum protein is
detected
 Certain antibody will only
attach to one species’s
serum protein

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 Methods
1. Ring Precipitin Test (Uhlenhut)
2. Ouchterlony Double Diffusion
3. Crossed-Over Electrophoresis

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 Ring Precipitin Test (1)
 Making Human Antiglobulin Serum

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Ring Precipitin Test (2)

Blood sample (dilute)

in top layer

Antiserum in bottom
layer

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Ring Precipitin Test (2)

Blood sample (dilute)

in top layer
Precipitate means blood &
antiserum species match
Antiserum in bottom
layer

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 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
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 Crossed-Over Electrophoresis

Antiserum Blood Stain

Gel

Holes

 Antiserum and Blood move toward each other

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 Crossed-Over Electrophoresis

Antiserum Blood Stain

 Antiserum and Blood move toward each other

 If line forms, antiserum and blood match

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 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

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 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

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 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

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ABO Markers (2)

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 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
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Direct Test

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 Indirect Test
(Absorption Elution)

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 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

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 Protein or Enzyme Markers (2)
+ Ladder Type 1 Type 2 Type 3

• Charge makes proteins

move through gel
• Different shapes move
at different rates
• After several minutes,
their location will tell
what type they are.
-
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Protein or Enzyme Markers (3)

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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
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ABO Mendelian Inheritance

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 Case (1)
Phenotypes
Phenotypes
Father Mother

A AB

Child

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 Case (2)
Phenotypes
Phenotypes
Father Mother

O AB

AB

Child

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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

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 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
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 Rape
• Serological evidence is crucial
• Forceful physical contact 
Transfer of evidence:
– Blood, Semen, Perspiration, Urine
– Hair and Nail
• Proving sexual intercourse by finding
semen

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 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

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 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
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 Semen Stains
 Visual: yellowish-white
 Tactile: stiff
 Odor: ~ chlorine
 ALS: fluorescence

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 + On
 Semen Analysis Steps

Presumptive Stain /
Chemical Analysis
Fluid?

+ - Semen (-)

Confirmatory Microscopic / EP / Immunological

- + Semen (+)

Blood Group
 Presumptive Tests
 Crystal Tests
 Florence
 Berberio
 Puranen
 Enzym Markers
 Seminal Acid Phosphatase
 Zinc Test

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 Florence Test
 Choline + lugol (KI)  Crystals of choline
periodide (brownish needle rhomboid shaped)

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 Berberio Test
 Spermine + saturated picric acid  Crystals of
spermine picrate (yellowish neddle shaped)

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 Puranen Test
 Spermine + Naphtol S Yellow  Crystals of
spermine flavinate (yellowish neddle shaped)

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 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

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 Zinc Test
 Zinc + Tris (hydroxymethyl) aminomethane +
PAN (1-(2-Pyridylazo)-2-naphthol)  Pink

 + On
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 + 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

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Direct Technique

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 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

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 Baecchi
 Sperm cells on clothing, bedding, etc
 Two dyes used:
 Acid Fuchsin
 Methylene Blue

 + On

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 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

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PSA Test by Electrophoresis

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 PSA Rapid Test Device
Membrane Based Immunochromatography

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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 -

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 If Semen, Whom Does it Belong to?
• Blood Group Type
– Secretor : ABO, Lewis Blood Typing
– Methods: Absorption Inhibition

• DNA : Y-STR
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 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
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 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.

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THANK YOU