Composition, function, collection, preservation and identification of blood, semen, saliva, urine, feces, sweat, tear, milk, menstrual blood and fetal blood;

1. FORENSIC SEROLOGY

2. Forensic Serology
 Detection
 Identification (which body fluid??? Species of origin of
sample???)
 Individualization of Body fluids

3. • 1863 ( 1st Presumptive test for
blood) The German scientist
Christian Schonbein discovers the
oxidation of hydrogen peroxide when
exposed to hemoglobin.
1900 (Serology ABO
Grouping): Karl Landsteiner
first discovers human blood
groups (the ABO system);he
is awarded the Nobel prize
for this in 1930.
1971(Standard protocol): Brian Culliford
publishes The Examination and Typing of
Bloodstains in the Crime Laboratory,
establishing protocols and standard
methods for typing of protein and enzyme
markers.

4. 1910 (Serology Department):
Serology Department’ was
established in Calcutta this institute
provided valuable scientific support
by analyzing biological materials for
crime investigations. After
independence, the department was
renamed as ‘Office of the Serologist
and Chemical Examiner to the
Government of India’.
1965 (2nd FSL):the second
central forensic science
laboratory was established at
Hyderabad. The CFSL,
Hyderabad initially established
analytical facilities in the
disciplines of Forensic Physics,
Forensic Chemistry and
Forensic Biology.
1957 (CFSL):The first Central Forensic
Science Laboratory was established at
Calcutta. laboratory was organized
into four basic disciplines viz. Forensic
Physics, Forensic Chemistry, Forensic
Biology and Forensic Ballistics.

5. Development of Forensic Serology
(1)Antigen polymorphism
(1)Protein polymorphism
Recent advancements in body fluid
identification/ Development of
Rapid On spot tests
This Photo by Unknown Author is licensed under CC BY

6. Forensic Significance of Blood
 Violent Crime (H/CA/SA/AR……)
 Pool, dry stain, washed stain, blood
print, bloody shoeprint, bloodstain
pattern, small droplets, traces of
blood on weapon.
 Locard Principle of exchange

10. A father hacked his daughter and son-in-law to death in Tamil Nadu’s Tuticorin
district. The man, identified as Muthukutty (50), hails from Veerapatti village near
Kovilpatti city in the Tuticorin district. His daughter Reshma (20) was a second-year
student at a college in Kovilpatti. The girl was in love with Manikaraj (26), a daily wage
laborer from the area. They had recently got married despite Muthukutty not
approving of their relationship.
Reshma's father, Muthukutty, strongly objected to his love. The couple reportedly got
married a few days ago and returned to the village only two days back. Muthukutty
had strongly opposed their marriage. Later, through the village panchayat, both of
them were allowed to stay in the village. But Muthukutty was furious with his
daughter. When Reshma and her husband Manikaraj were alone at home last evening,
Muthukutty went there and killed both of them to death with a sickle. He later fled the
scene of the crime. Based on information about the incident, officials from
Ettayapuram police station went to the spot, recovered the bodies, and sent them to
Tuticorin Government Hospital for post-mortem. The police also later nabbed
Muthukutty, and further investigations are underway.
Man hacks daughter, son-in-law to death with a sickle in Tamil
Nadu over love marriage, arrested
Police officials in Tuticorin’s Ettayapuram arrested a 50-year-old man, Muthukutty, for hacking his daughter and son-in-law with a sickle after
they got married without his approval.
ADVERTISEMENT
Akshaya Nath
Tuticorin
July 26, 2022
UPDATED: July 26, 2022, 13:41 IST

11. IDENTIFY THE FOLLOWING
1)VICTIM(S)
2)CULPRIT (S)
3) CRIME
4)EVIDENCES WITH BLOOD/BLOODSTAIN
REVCOVRED FROM CRIME SCENE:
5)ADDITIONAL BLOOD SAMPLE(S):

12. Collection of Blood Evidence
DRY
Swab, cutting, scraping,
lifting, collect the entire
surface.
WET
REFERENCE

14. Presumptive Assays for Blood Identification
HEMOGLOBIN HEME= Ferro (fe2+) Protoporphyrin IX.

15. Presumptive Assays for Identification
• oxidation–reduction reaction catalyzed by the heme moiety of
the hemoglobin
 chemiluminescence, fluorescence, or colorimetric
 10–5−10–6-fold dilutions.
 positive reaction indicates the possible presence of blood

16. Colorimetric Assays
• Phenolphthalin Assay
• Leucomalachite Green (LMG) Assay
• Benzidine and Derivatives

17. Benzidine test
Benzidine Test - YouTube

21. Phenolphthalein test

23. Chemiluminescence and Fluorescence Assays
• Luminol (3-Aminophthalhydrazide)
• Fluorescin

24. Advantage/Disadvantage:

25.  425– 485 nm using an alternate light source device
 Advantage

27. Factors Affecting Presumptive Assay Results
false-positive reaction in
presence of Strong oxidants
 metal salts, such as copper and
nickel salts,
 household bleaches and
cleaners (hypochlorite ions)
 and hair-coloring products
(hydrogen peroxide)
 Solution
Horseradish (plant peroxidase)
Solution
Strong Reductants:
metal ions including lithium and zinc (Rare)

28. Takayama test
Confirmatory Assays for Identification
Microcrystal Assays

29. Takayama test
 It was first developed in 1912 by Masaeo Takayama, a Japanese forensic
pathologist. Later, Takayama’s name has become the synonym of
hemochromogen crystals.
 The principle is based on the formation of hemochromogen (or called pyridine
ferroprotoporphyrin) with a reaction with pyridine.
 In general, heme has six bonding sites of which four are attached to classic
nitrogen bonding. While the other two sites also have N bonding but with the
organic base of pyridine. In addition, the central iron atom has a 2+ charge i.e.
Fe2+.
 This altogether makes a hexa-coordination complex which has feathery pink
structures that are known to be Takayama crystals.

30. Takayama test

31. Confirmatory Assays for Identification
Microcrystal Assays
Hemochromagen Crystal Assay/Takayama
pyridine ferroprotoporphyrin

32. Teichman test

33. Teichman test
 Teichmann is a microcrystal confirmatory test for blood, used by forensic experts. It is
also called Hemin or Hematin test.
 The test was first developed by Ludwik Karol Teichmann, a Polish anatomist in 1853.
He documented these microcrystals in his paper of 1853 and named them
‘Hematins’. But later, these crystals called to be Teichmann’s crystals.
 The test is based on the reaction of the heme part of blood with potassium halide
(Bromide, iodide, chloride) and glacial acetic acid to form brown-colored rhombic
crystals. These prismatic-rhombic-shaped brown crystals are a sign of the formation
of hematin chloride (ferriprotoporphyrin chloride) crystals.
 Generally, a Hemoglobin has six binding non-proteinous sites. Four have nitrogen
coordination bonding to tetrapyrrole ring with iron (Fe2+). While the fifth bonding is
between the iron atom and nitrogen of the deprotonated proximal histidine residue.
Lastly, it either has a water or oxygen bonding (oxygenated Hb).
 When Potassium halide with glacial acetic acid reacts with heme, a heme derivative
known as Hematin is formed with a central iron in the ferric state (Fe3+).

34. Teichman test
Potassium
Halide

35. Confirmatory Assays for Identification
Microcrystal Assays
Hematin Crystal Assay
ferriprotoporphyrin chloride

36. Spectroscopy test
Reagent:
Solution # 1: 0.2% Sodium lauryl sulphate in water Solution # 2:
0.2% Mercaptoethanol in 1% NH3 solution
Steps
I. Bloodstain + solution 1 (incubate at 37 degree celius for 15-
20 minutes)
II. Add solution 2 (after for 5-10 minus) Scan from 500-600nm.
Lambda max at 558 nm and 529 nm, indicate the presence of
haemoglobin derivatives.
Standards and Controls: Known bloodstains of various ages must
be tested, Oxyhaemoglobin exhibits absorption peak at 576 and
538 nm. The apparent shift is thought to be due to the formation
of reduced haemoglobin derivatives

37. Lateral Immunoassays

38. Immunoassays for hemoglobin
The SERATEC® HemDirect Hemoglobin Test :
 serves the rapid identification of human blood for forensic
purposes.
 The result is interpreted visually by the appearance of a red
test line in hemoglobin positive samples.
 The test can be used in the lab or directly in the field.
 Sensitivity: cut-off at 20 ng/mL Hemoglobin
 no special training necessary
 compatible with DNA extraction and typing

39. Hexagon OBTI
• The test detects whole blood up to a dilution of 1 :
2,000,000. As little as 250 red cells are required for a
positive result.
• detecting 0.05 µg/ml hemoglobin Human hemoglobin (hHb)
in the sample
• False positive: They were obtained with blood from
primates (gorilla, and of some Mustelidae (weasel, badger).
• The Blood of the following animals did not react with the
Hexagon OBTI: cattle, pig, sheep, goat, horse, rabbit,
chicken, duck,goose, turkey, guinea pig, red deer, cat, dog.

40. • The ABAcard® HemaTrace®
• The presence of only one pink line in area
• This test has shown that false positives may occur with
ferret blood.

41. Immunoassays for Glycophorin A
• Rapid Stain Identification of Human Blood (RSID™-
Blood)
• No cross-reactivity with human saliva, semen, breast
milk, amniotic fluid, vaginal fluid or urine has been
observed.
• No cross reactivity with animal blood has been
observed.
• Species tested: ferret, skunk, opossum, dog, cat, cow,
pig, chicken, owl, horse, goat, turtle, elk, deer, tiger,
alpaca, orangutan, gorilla, spider monkey, bonobo,
and baboon.
• Test Sensitivity The sensitivity for RSID™-Blood, used
as suggested, is less than < 1 µl of human blood.

42. Source: Sensabaugh 2016

46. RT PCR Assays
HBA1 (Hemoglobin Subunit Alpha 1) is a Protein Coding
gene (located at 16p13.3)
SPTB (Beta Spectrin) encodes a member of the spectrin gene family.
Spectrin proteins, along with ankyrin, play a role in cell membrane
organization and stability of erythrocyte membranes

47. HMBS Gene (hydroxymethylbilane synthase superfamily).
The encoded protein is the third enzyme of the heme
biosynthetic pathway and catalyzes the head-to-tail
condensation of four porphobilinogen molecules into the
linear hydroxymethylbilane

48. Fetal blood

49. *The production of fetal Hb is switched off after the birth.
Hb Adult Hb Fetal
Structure 2 alpha and 2 beta chains 2 alpha and 2 gamma chains
Lifespan 120 days 80 days
Oxygen carrying capacity Less oxygen carrying capacity More oxygen carrying capacity

50. Fetal blood
• Tests for fetal blood
• The test is known as APT test or
Alkali denaturation test.
• Blood cells are first lysed. SO
distilled water is added to the stain
and the hemoglobin is extracted
out of the RBCs.
• To the lysed Hemoglobin, 1%NaOH
is added and left to stand in room
temperature.
• Fetal hemoglobin will stay pink,
adult hemoglobin will turn yellow-
brown.

52. platelet activation aggregation to form platelet plugs at the
site of injury
Thrombin a serine protease, converts soluble fibrinogen
into fibrin
Fibrin, aggregates with the platelet plugs and leads to the
cessation of bleeding by forming blood clots called thrombi
. Blood clots are prevented from accumulating during
menstruation by forming low amounts of platelet plugs
and synthesizing coagulation factor inhibitors that inhibit
blood coagulation.
Additionally, fibrinolysis is activated, during which
thrombus is broken down by a protease known as
plasmin.

53. D-dimer Assay

55. Menstrual blood
• Menstrual blood is the product of menstruation in
menstruating women.
• On an average 35 ml of menstrual blood is secreted by
the female reproductive system during a single
menstruation cycle.
• Menstruation is the regular discharge of blood and
mucosal tissue from inner lining of the uterus.
• It must be noted that a woman starts menstruating
around 12-15 years of age (menarche) and stops
menstruating around 45-55 years of age (menopause).
• Menstrual blood differs from human circulatory blood in
terms of its content and its properties.

56. Menstrual blood

57. MB PB

58. Pale yellow

59. RNA based Assays
• Matrix metalloproteinase (MMP) genes are considered tissue-specific
markers for human endometrium tissues.
• MMPs are zinc-dependent endopeptidases that degrade extracellular matrix
components.
• The most commonly used markers for the forensic identification of menstrual
blood are MMP7 and MMP11
• Both MMP7 and MMP11 mRNA expressions are elevated at the menstrual
phase and remain at high levels during the proliferative phase.
• It is also known that MMPs’ mRNA may be elevated in postpartum, wound
healing, and metastatic cancer conditions, which may potentially lead to a
false-positive identification of menstrual blood.

60. Semen • What is semen?
• Forensic significance?

61. Semen (On an average men secrete 3.4ml of semen per
ejaculation. The principle component – spermatozoa
and seminal fluid.
Seminal fluid: seminal vesicle
(65-75%), prostate gland (25-
30%) and cowper’s gland/
bulbourethral gland (0-1%).
The testes (testicles) which is made up
of seminiferous tubules (sertoli cells)
and connected to the penis by the vas
deferens.

63. Lighting Techniques for Visual Examination of Semen Stains
Presumptive Assays (Acid Phosphatase Techniques via Colorimetric
Assays/ Flurometric Assays )
Confirmatory Assays
1.Microscopic Examination of Spermatozoa
2.Crystal test (Choline and Barberios Test)
3.Identification of Prostate-Specific Antigen
(Immunochromatographic Assays)
4.Identification of Seminal Vesicle–Specific Antigen
(Immunochromatographic Assays)
5.RNA-Based Assays
Analytical Techniques for Identifying Semen

64. 450-495nm
Limitations:
1. False positive for other bodily fluid
stains, such as saliva and urine
stains.
2. The intensity of the fluorescence
can be affected by different colors
of substrates, and the material,
such as clothing, where semen
stains have been deposited

65. Acid
phosphat
ase (AP)
• phosphatases with optimal activity in an acidic pH environment.
• prostate-derived AP contributes most of the AP activity present in semen.
• Human seminal plasma at levels100–1000 higher than in any other body fluid i.e.
vaginal fluids .
• AP levels in semen are not affected by vasectomies.
• AP half-life of AP activity at 7°C is 6 months. However, the half-life is decreased if
a sample is stored in a wet environment.
• AP activity can be detected from dry seminal stains stored at –20°C up to 1 year.
• Tests: Colorimetric assay and Fluorometric assays (4-Methylumbelliferone
phosphate (MUP)

68. fluorescence under ultraviolet light.

69. STAINS
Nuclear Fast Red (NFR), Picroindigocarmine
(PIC)
The acrosomal cap and the nucleus stain pink-red
the sperm tails and the midpiece stain blue-green.
 a small portion of a stain with
water, followed by gentle
vortexing. The suspension is
then transferred to a slide and
evaporated at room
temperature or fixed with low
heat.
 Alternatively, it can be
transferred by dampening the
stain with water and rubbing or
rolling it onto a microscope
slide.

71. Dark brown colored crystal of Choline Iodide
Florence Test

72. Barberio’s Test
Needle shaped yellow color crystal of spermine picrate

73.  SERATEC® PSA Semiquant is an
immunochromatographic rapid
test for the detection of the
Prostate-specific Antigen (PSA)
 High sensitivity: cut-off at 0.5
ng/mL of PSA
Prostate-Specific Antigen

74. ABA card® P30
Prostate-Specific
Antigen

75. •RSID™-Semen (Immuno-Chromatographic Lateral
Flow Strip Test)
 Specific for human semenogelin antigen
 Other human body fluids do NOT cross-react with this
test procedure. (Fluids tested: saliva, blood, urine,
vaginal secretions, menstrual blood)
 Animal seminal fluid does NOT cross-react with test
procedure. (Samples tested: bovine, porcine,
caprine, and ovine semen)
 Detect as Little as 1 µL of Human Seminal Fluid
Seminal Vesicle–Specific Antigen

78.  Saliva traces recovered from such diverse sources as cigarette
butts, chewed food,and drinking containers have yielded
complete DNA profiles.
 Of particular note, many sexual assaults include oral contact on
the breasts, neck, and around the mouth; DNA profiles obtained
from these sites may be probative.
 Bitemarks
 Saliva also contains buccal cells, allowing the possibility of DNA
profiling.
SALIVA AS EVIDENCE

80. Visual Examination:
Presumptive Assays
1.Buccal epithelium cells
2. Starch Iodine Assay
3.Colorimetric Assays i.e.Phadebas pressTest, SALIgAE® kit (Abacus
Diagnostics
Confirmatory Assays
1.Identification of Human salivary amylase
(Immunochromatographic i.e. RSID®-Saliva kit )
2. RNA-Based Assays
Analytical Techniques for Identifying Saliva

81. 470 (Bluish Florescence)

82. Buccal epithelium cells stained with methylene
Blue

83. Starch Iodine Assay

84. Phadebas® Forensic Press Test

85. SALIgAE® kit (Abacus Diagnostics
1. Place approximately 5 mm2 cutting or 1 /2 of a
swab into a sterile 1.5 ml microcentrifuge tube.
2. Pipette 30 µl – 50 µl of sterile deionized water
into the tube.
3. Incubate for 30 minutes at room temperature.
4. Allow the SALIgAE® kit test vials to warm to
room temperature
5. Add 8 µl of sample to the test vial
6. Mix gently

86. •Lateral flow strip test
•Animal saliva does not cross
react with this test procedure
•No cross reaction observed with
blood, urine, semen, vaginal
secretions, or menstrual blood.
•Specific for human salivary -
Amylase Antigen
•Detection limit upto 1 μL
RSID®-Saliva kit

91. Presumptive assays
Urea
• DMAC Assay (colorimetric and florimetric)
• Microscopic crystal assay
• Urease assay
• Chromatography GC-MS, Thin layer
chromatography
Creatinine
• Colorimetric assay Jaffe test
• Chromatographic assay GC-MS
Uric acid
• Uricase assay
Confirmatory
assay
• Tamm-Horsfall
glycoprotein
• 17-ketosteriods
*Other bodily fluids, such as sweat, also contain these chemical components

92. Urine
• UV examination - Urine stains fluoresce as
yellow/pale blue in UV light.
• Flame test - On heating gently over a flame, the
characteristic odour of urine may be detected.

93. para-dimethylaminocinnamaldehyde
(DMAC) assay
light source at 473–548 nm
Limitations

94. Urine Microcrystal test
• Urea Nitrate Crystal test - An aqueous
extract of the stain when reacted with
one drop of conc. Nitric acid on a slide
forms hexagonal urea nitrate crystals.

95. Urease activity
The ammonia is detected using an acid-base indicator, bromthymol blue, which
exhibits a blue color. Alternatively, the ammonia can be detected by manganese
and silver nitrates, which exhibit a black color.

96. • Creatinine test (Modified Jaffe’s test) – One drop of picric acid is added to stain
followed by 5% Sodium hydroxide – Brown/orange color shows presence of
creatinine.

97. Measuring the disappearance of the absorption of
uric acid at 293 nm after treatment with uricase that
catalyzes the oxidation of uric acid to 5-
hydroxyisourate

98. Confirmatory test for Urine
 RSID™-Urine is a lateral flow immunochromatographic strip test
designed to detect the presence of the TammHorsfall (THP)
glycoprotein (sometimes called uromodulin).
 Tamm-Horsfall is the most abundant protein present in urine. It is
secreted by the thick ascending limb of the loop of Henle, and
then excreted into urine at a rate of 80-200 mg/day
 RSID™-Urine is specific for urine and does not crossreact with any
other human bodily fluids.

99. Confirmatory test for Urine :
17-Ketosteroids using LC-MS
metabolite of
testosterone and
dihydrotestosterone
endogenous steroid hormone
precursor
metabolite of testosterone

100. Fecal Matter
• Sodomy
• assault with fecal matter
• Vandalism
• burglary during which the perpetrator defecated at the scene

101. Human feces contain undigested foodstuffs, sloughed intestinal epithelial
cells, intestinal bacteria, bile pigments, electrolytes, and water.

102. Forensic Examination
Macroscopic Examination
Microscopic Examination
Urobilinoid Test
Fecal bacterial Identification

103.  The normal brown color of feces primarily results from the
presence of urobilinoids, which are heme catabolic by-products.
 The characteristic odor of feces is caused by the metabolic by-
products of the intestinal bacterial flora. Indole, skatole, and
hydrogen sulfide are the compounds that are responsible for the
odor of feces
Feces Macroscopic examination

104. Feces Microscopic examination
• Fecal matter can be transferred from samples of clothing by
scraping with a sterile stainless steel spatula. The fecal matter is
then hydrated in 6% formalin solution for 1-2 days prior to
microscopic examination.
• A small of amount of stain scraping is mounted on a slide with a
drop of Lugol’s Iodine and observed microscopically for
undigested vegetative matter, muscle fibres, etc
• The presence of characteristic undigested foodstuffs can indicate
human feces.

105. Urobilinoids Tests
Albumin
glucuronic acid
Bile
Unconjugation of BG
IB
Oxidation by IB Oxidation by IB

106. Schlesinger and Edelman tests
Schlesinger test
Sample
+
saturated zinc acetate
(1% zinc acetate methoxyethanol solution and
0.2% Tris)
urobilinoid– zinc chelation complex
(green fluorescence under UV at 507
and 514 nm)
Edelman test
Sample
+
mercuric salt solution
a pink-colored compound
+
Zinc Salt
(fluorescence under UV)
sonicated for 5 min, heated at 100°C
for 10 min, cooled, and centrifuged
Limitation: ST vs. ET
Low Species specificity

107. Fecal Bacterial Identification

108. Fecal Bacterial Identification
30% of fecal microbiota comprises of Bacteroides (rod-shaped,
anaerobic gram-negative bacteria that digests complex
carbohydrates and other substances that cannot be digested by
human enzymes.)
B. uniformis
is not detectable in blood, saliva, semen, urine, vaginal
fluids, or on skin surfaces.
considered as a specific indicator bacterium for forensic
fecal identification.
B. vulgatus c
an also be detected in vaginal fluid samples
Limitations:
 Species non specific
 DIET dependent (Bacteriods rich in
saturated fats and protein rich diet )

109. sweat

110. Presumptive test: Lactate, lactic acid, urea, and single amino acids
Scanning Electron Microscope EDX

111. Presumptive test: Lactate, lactic acid, urea, and single
amino acids using Raman Microscpectroscopy

112. Confirmatory assay for dermcidin
• a class of human antimicrobial peptides of the
innate immune defense system and plays an
important role in protecting epithelial barriers
from infections.
• expressed in eccrine sweat glands
• The detection can be performed using ELISA assays
utilizing antibodies specific to human dermcidin.
(highly sensitive for 10,000-fold dilution).
• Dermcidin is encoded by the DCD gene. Its mRNA
can be detected using reverse transcription
polymerase chain reaction (RT-PCR) assays

114. Tear
Basal
Tears
Coats the
superficial layer
of eye
Reflex
Tears
Secreted in
response to
external stimuli
Psychic
Tears
Secreted due to
emotional and
cognitive
It is made up of water, electrolytes,
proteins and mucins. The ratio of each
component varies at different intervals in
the human body.

115. Tear • Tests for tears
• Lactoferrin is the target molecule.
• Lactoferrin (LF), also known as lactotransferrin (LTF),
is a multifunctional protein of the transferrin family.
Lactoferrin is a globular glycoprotein with a
molecular mass of about 80 kDa
• Specific testing kits with patented tech are available
for testing for Lactoferrin.

117. Milk • Milk (Lactation) is a nutrient-rich liquid secreted by the
mammary glands (breasts) of women to nourish their
offspring (child).
• The chief function of milk secretion is to provide nutrition. It
also provides immunity, emotional connection etc.
• Milk contains nutrients, proteins and lactose.
• The first milk produced after childbirth is known as
colostrum and contains antibodies in addition to the
above.
• Human milk and animal milk differs in certain means – PUFA
lipoproteins and Vitamin D are present in human milk and
absent in animal milk.

118. Milk
• Tests for milk
• Lactose is a target molecule – Reagent test kits
• Vitamin D, LDLs can also be looked for using
biochemical analysis.