Forensic Serology

1. FORENSIC SEROLOGY

2. • 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.

3. 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.

4. 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

5. 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

9. 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

10. 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):

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

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

14. 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

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

16. Benzidine test
Benzidine Test - YouTube

20. Phenolphthalein test

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

23. Advantage/Disadvantage:

24.  425– 485 nm using an alternate light source device
 Advantage

26. 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)

27. Takayama test
Confirmatory Assays for Identification
Microcrystal Assays

28. 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.

29. Takayama test

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

31. Teichman test

32. 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+).

33. Teichman test
Potassium
Halide

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

35. 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

36. Lateral Immunoassays

37. 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

38. 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.

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

40. 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.

41. Source: Sensabaugh 2016

42. 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

43. 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

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

45. 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.

47. 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

48. 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

49. 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)

52. fluorescence under ultraviolet light.

53. 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.

55. Dark brown colored crystal of Choline Iodide
Florence Test

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

57.  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

58. ABA card® P30
Prostate-Specific
Antigen

59. •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

62.  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

64. 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

65. 470 (Bluish Florescence)

66. Buccal epithelium cells stained with methylene
Blue

67. Starch Iodine Assay

68. Phadebas® Forensic Press Test

69. 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

70. •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