FMT-PPT

1. TRACE EVIDENCETRACE EVIDENCE

2. “Microscopic materials
recovered as evidence that
is used to help/solve
criminal cases”

3. • Edmund Locard - exchange principle
“Every contact leaves a trace “
• Trace evidence analysis: The
characterization, identification &
comparison of microscopic materials in
criminal cases.
• Trace evidence helps to crimes by linking
people, places & things involved in a crime

4. • Traces become evidence when introduced
into the court
• At the onset of an investigation to develop
a suspect

5. • Most commonly encountered trace
evidences
- Hair, fiber, paint, glass, plants, wood, seed,
fungal spores, pollen grains, insects, soil,
sand, mineral particles, feathers, metal
particles, gunshot residues, explosives,
adhesives, plastics, lubricants, cosmetics,
finger prints, foot prints, lip prints, bite
marks, blood, seminal, salivary, sweat,
vaginal secretions, anal secretions, C S F
stains etc

6. what is the Purpose of trace evidence
analysis ?
• To establish identification of criminal/victim
• To exclude the person from the list of
suspects in crime
• To connect to the scene of crime
• To connect to the weapon & crime
• At the onset of an investigation to develop a
suspect/group of suspects

7. Blood & Bloodstains
• Handling
• Packing
• Storage
• Examination
Blood stained knife

8. • Any stains sent may/may not be a blood, to confirm
submit for examination
1.Is it blood/not
2.If blood – belong to human/animal
3.If human – from living/dead
from artery/vein
from which part of body
how old
age of person
sex
Blood group
Belong to victim/assailant

9. Above queries can be answered by:
• Physical examination
• Physico – chemical test
• Chemical examination
• Microscopic examination
• Micro-chemical test
• Spectroscopic examination
• Serological – immunological studies
• Enzymological studies

10. Physical Examination
• Natural light
• Color
• Unclear/invisible stains- 230-269 nm
frequency UV light

11. Examination of Blood stains on floor

14. Physico – chemical test: To differentiate
the stain is due to blood/vegetables/dyes
etc
• Vegetable stains give green color with
ammonia
• Aniline dyes will give yellow color with
nitric acid
• Blood stains will remain unchanged with
both of the above

15. Presumptive Test (Chemical examination)
1. Tetramethyl Benzidine (TMB) Test
Cutting or swabbing of the stain on filter
paper + drop of TMB Solution + drop of
3% H2O2
Blue-green color

16. 2. Phenolphthalein Test (Kastle-Meyer Test)
Cutting or swabbing of the stain on
filter paper + 2-3 drops of Ethanol + 2 drops
phenolphthalein soln
+ 2-3 drops of 3% H2O2
Pink color

17. Microscopic Examination:
Put a drop of saline on stain/clot after
placing it on glass slide
Mix well Observe under
wait for 30 min various magnification
RBCs if circular, biconvex & non-
nucleated(human), circular/oval &
nucleated(amphibian)

19. Confirmatory Test (micro-chemical tests)
1. Takayama Test (Haemochromogen Crystal
Test)
Put a material on microscopic slide cover with
a cover slip + a drop of Takayama Reagent
allow to flow under cover slip
Warm on plate 65o
C
for 10-20 seconds
Cool
Observe under 100X
Pink needle shaped crystals of pyridine
hemochromogen

20. 2. Teichmann’s Test(Haemin Crystal Test)
Place material on slide and put a cover
slip + a drop of Teichmann’s Reagent allow
to flow under cover slip
Warm on plate
65o
C
for 10-20 seconds
Cool
Observe under
100X
Rhombohedron shaped crystals of
ferroprotoprophyrin chloride

21. Spectroscopic Examination:
• When light spectrum is viewed through
blood, certain colors are absorbed
• The absorbed color appear as dark
bands called absorption bands & full
spectrum is called absorption spectrum
• Microspectroscope is used to observe
this

23. Artery or vein:
• Arterial blood will be scattered
• venous blood will not

24. Blood group: Commonly employed blood
groups are ABO & Rh systems
• Done by routine methods using antiserum
• If the blood recovered as a clot/stain on
any particle the grouping can be done by
using absorption-elution technique

25. M L Importance
• Disputed paternity
- Child born under lawful marriage but the
husband denies that he is not the father of
child
- Child born out of lawful marriage & mother
accuses certain man as a father but man
denies the accusation

26. - Women adopt a child claiming it as her
own in order to obtain a share in husbands
property
- In suits of nullity of marriage
• Disputed maternity
- Same child claimed by two women
- Exchanged babies in maternity
home/hospitals

27. • Blood groups commonly used in these
cases are- ABO, MNS, Rh, Kell, Lutheran,
Duffy, Kidd etc
• Limitations- They may exclude a person
as a possible father/mother but they
cannot definitely establish
paternity/maternity
• DNA finger printing will positively fix the
paternity/maternity

28. • Identity
- In unidentified bodies by blood grouping &
DNA analysis

29. • Crimes
- Blood stains on clothing of an suspect
- Blood stains on weapons
- Blood stains on particular place
- Blood stains on vehicles

30. • Cause of death
- Mismatched blood transfusion
- Poisons can be detected in blood
Eg- alcohol, organophosphorus
compounds etc

31. SEMINAL
FLUID

33. SEMINAL FLUID
Introduction
• Semen is a body fluid present in human
males.
• It is a viscid mucilaginous fluid with faint
yellow colour and characteristic odour
called seminal odour.
• Volume is about 2-5 ml per ejaculate.
• No. of Spermatozoa= 60-150 million per
ml.
• PH-7.4

34. COMPOSITION
• Semen consists of the following
1. Spermatozoa (9%)
2. Seminal Plasma (90%)
3. Epithelial Cell (1%)
Major part of seminal plasma is derived from
secretions of seminal vesicles & prostate.
1.Secretions from seminal vesicles include-:
Fructose, Phosphorylcholine,
Prostaglandins.

35. 2. Secretions from prostate include-:
Acid phosphatase, Spermine, Citric acid,
Cholesterol, Phospholipids, Fibrinolysin,
Zinc.

36. Methods Applied for Detection of
Seminal Stains
• Physical Examination
• Chemical Examination
• Microscopic Examination

37. Physical examination
Seminal stains when dry have a grayish-
white or yellow-grey colour and show an
irregular, map like outline.
The cloth is stiffened as if starched.
 A fresh stain on a non absorbent material
appears translucent.
When examined under UV light they show
a fluorescence of a bluish-white colour.

38. Chemical examination
The tests used to detect Seminal Stains are:
 Florence Test
 Barberio Test
Acid Phosphatase Test
Creatine phosphokinase
Immunological method

39. Florence Test
Basis: Choline is detected in this method.
If semen is present - dark brown rhombic shaped
crystals arranged in clusters, rosettes etc. of
choline iodide appear immediately.

40. Creatinine phophokinase
Bases: Detection of creatinine phosphokinase.
Normal seminal fluid content - 385 - 14000 U of
CPK/W.
Diagnosis: >400 U of CPK / ml.
Immunological method
PSA is a glycoprotein & is found in seminal plasma,
male urine & blood but not in females. It is found in
vaginal fluid upto 27hrs after sexual intercourse.

41. Barberio’s test
Basis: Detection of Spermin
Procedure: A few drops of Barberio’s reagent when
added to spermatic fluid produces crystals of sperm
in picrate (needle shaped, rhombic & of yellow
colour).

42. Microscopic examination
1. Small piece of stained fabric is moistened with few
drops of 1% HCl or 2-3% acetic acid in a watch
glass-for ½ -1hr when stains are fresh or 2-4hrs
when old.
2. The slide is stained with methylene blue for 1-30
min & counter stained with eosin for 2 min.

44. MOTILITY OF SPERMS
At room temp.- full motility-: 3hrs
50% motile-: 8hrs
10% motile-: 24hrs
Complete sperms are found only upto 24 hrs and
then they separate into heads & tails which can
be identified upto 4 days.

45. PROOF OF SEMEN
 The only absolute proof is the finding of atleast one
unbroken spermatozoan or electrophoretic LHD
isoenzyme detection of sperms.

46. MEDICOLEGAL SIGNIFICANCE
Rape
Sodomy (Anal intercourse)
Bestiality (Sexual intercourse by a human being
with a lower animal like dogs, calves, sheep etc.)
In case of false Accusation by a women
 Incest (Sexual intercourse in blood relation) and
 Sexual Murders.

47. Saliva and Salivary Stains
• Cigarettes/bidi ends
• Apparels
• Bite marks
Bite mark

48. • Starch- Iodine Test
5 x 5 mm of sample similar unstained control piece similar known saliva stain
3 drops of soluble starch soln
Mix & cork incubate for 1 hour at 37o
C
2 drops of Lugol’s iodine
Dark blue starch-iodine complex should
be observed in the 2nd
& 3rd
tubes

49. • Blood grouping - in case of secretors
(Persons who secrete blood group
antigens in saliva)
• Sex determination - If epithelial cells are
present in the saliva/salivary stains - Barr
body detection

50. M L Importance
• Identification of victim/criminal (sex, blood
group etc) from cigarettes/bidi ends, bite
marks, apparels like bottles, spoons etc
- Recovered at the scene of crime
- Swabs collected from the bite marks in
sexual assaults

51. TRACE EVIDENCETRACE EVIDENCE

52. Semen & Seminal Stains
• Handling
• Packing
• Storage
• Examination

53. Type of sample At the scene Preferred packing Comments
Wet staining
Portable items
Fixed surface
Remove item.
Cut out stained area
(+control unstained
area) /remove on
cotton wool swab
(+control swab)
Paper bag
Polythene bag/bottle
Standard swab tube.
Freeze.
Allow to dry in place
if possible.
Package controls
separately from
stains.
Dry staining
Portable items
Fixed surface
Vegetation stained
with semen
Soil stained with
semen
Remove item.
Cut out stained area
(+control unstained
area) /scrape with a
new scalpel into
container.
Remove stained
portion.
Remove layer of soil
onto tray
Paper bag
Polythene bag/bottle
Bottle/Perspex box.
Paper bag
On tray in paper bag.
Store at room
temperature.
Pack controls
separately.
Dry & store at room
temperature or
refrigerate.
Submit at once.
Refrigerate
meantime.

54. • Physical Examination
• Color - Thick, yellowish white, glairy,
opalescent
• Odor - Seminal
• Texture - Starchy
• Appearance - Preliminary examination
under filtered UV light. The fluorescence
of the seminal stains is of a bluish white
color

55. Presumptive Test
• Florence Test
• Stain is extracted by 10% HCl
• Extracted drop is placed on a glass slide
& allowed dry
• Place a cover slip over it & allow a drop
of Florence solution to run under cover
slip
• If semen is present dark brown crystals
of choline iodide appear immediately

56. • Barberios test
• Take the fluid/extracted stain on a glass
slide
• Add few drops of aqueous/alcoholic
solution of picric acid
• Spermatic fluid produces yellow needle
shaped rhombic crystals of spermine
picrate.

57. • Acid Phosphatase Test
2 x 2 mm of suspected seminal stain on
Whatman filter paper + 1-2 drops of Step
1 Reagent
allow for 30 seconds No color should develop
1drop of Step 2 Reagent
after 10 seconds
purple color, indicative of semen

58. Reagent Preparations:
Buffer
Glacial Acetic acid
Sodium acetate anhydrous
Distilled water
Step 1 Reagent
Buffer
Sodium alpha-naphthyl Phosphate,
0.25% (w/v)
Step 2 Reagent
Buffer
Naphthanil diazo blue B, 0.5% (w/v)
1 ml
2 g
100 ml
50 ml
126mg
50 ml
250mg

59. • Creatine Phosphokinase Test:
Test detects the presence of Creatine
Phosphokinase enzyme which is more
then doubles in sperm as compared to
other body fluids

60. Confirmatory Test
• Microscopic Examination
• 1 cm2
of stain + few drops of acidulated
water
Agitate by flicking the tube
withdraw the liquid with a pipette
& centrifuge for 30 sec
withdraw the supernatant
Collect insoluble components on slide
Fix in dilute H2SO4 acid and dry

61. • Gram Staining
Fix the smear to the slide by gentle heating
Add Crystal violet to slide for 1min
Rinse with tap water
Gram’s iodine to slide for 1min
Rinse with tap water
Add 95% ethanol and tilt the slide
back and forth 10 sec
Rinse with tap water
Safranin Soln
for 1min
Air dry. Examine with oil immersion at 100X.

62. •Spermatozoa appear as purple bodies, oval in
shape with a clearly discernible acrosomal
cap.

65. • Grading of Spermatozoa on Smear
- Few; difficult to locate – 1
- Some in some fields – 2
- Some in many fields; easy to locate – 3
- Many in most fields - 4

67. Electrophoretic Methods
Acid Phosphatase:
• Polyacrylamide gel electrophoresis
followed by staining with methyl
umbelliferyl phosphate reagent
• Distinguish from the acid phosphatase of
other substances
• Superior to LDH- semen identified even in
absence of sperms

68. Lactate dehydrogenase:
• Sperm specific LDH can be seperated
from other LDH isoenzyme of semen by
polyacrylamide gel electrophoresis
- In stain over 4 weeks
- Pattern is different from other animals
- More specific than microscopy
- Cannot be used in azoospermic &
vasectomised individuals

69. M L Importance
• Civil cases
- Disputed paternity
- Legitimacy
- Artificial insemination
- Divorce cases

70. - Compensation on failure of vasectomy,
leading to pregnancy
• Crimes
- Identification of accused in cases of sexual
assaults (Rape, sodomy etc)

71. Vomit
• Examination of vomit, presence of the
following accounted
- Mucus
- Free HCl
- Endothelial cells
- Undigested and semi digested food

72. • Test for Mucus
Extract
Add 33% acetic acid drop by drop
Opalescence appears
Add of more acetic acid
Opalescent does not disappear
Presence of mucus is confirmed

73. • Test for Free HCl (Gunzberg’s Test)
Suspected extract
+
1-2 drops of Gunzberg’s reagent & mix
Allowed to dry
Brilliant red color indicates HCl

74. • Endothelial Cells
Centrifuging the extract for 10 minutes
Thin film is made on a slide.
Endothelial Cells are observed under
microscope.

75. M L Importance
• Identification – DNA analysis of the
endothelial cells of vomitus
• Sex determination – Barr body
examination in these endothelial cells
• Vomitus on a particular place – connect
the person with the scene of crime

76. Urine Stains
• Physical Examination - Pale yellow or
pale blue fluoresce under UV light
• Odour Test – Ammonia odour

77. • Urea Nitrate Crystal Test - Add a drop
of conc HNO3 and cover hexagonal
stacked crystals of Urea nitrate seen
• Creatinine Test - stain on filter paper,
add a drop of picric acid followed by a
drop of 5% NaOH, Brown/orange
color

78. M L Importance
• Identification – DNA analysis of the
endothelial cells of urine stain
• Sex determination – Barr body
examination in these endothelial cells
• Urine stains on a particular place –
connect the person with the scene of
crime

79. Faecal Matter and Faecal Stain
• Physical Appearance - Brown in color due
to urobilinogen
• Microscopic Examination - A drop of
Lugol’s iodine is added & examined under
microscope
• Urobilinogen Test - Green fluorescent
zinc-urobilin complex

80. M L Importance
• Identification – DNA analysis of the
endothelial cells of faecal Stain
• Sex determination – Barr body
examination in these endothelial cells
• Identification of accused/victim in sodomy
• Faecal stains on a particular place –
connect the person with the scene of
crime

81. Vaginal Fluid & Stains of Vaginal
Secretions
• Physical Examination - Vaginal
Secretions, appear stiff on feeling when
these are on clothing
• Under UV light examination, these show
fluorescence.

82. • LUGOL’S Stain
Make & Fix the smear by gentle heating
Cover smear with Lugol’s iodine soln
. Add an
equal vol of water to the smear
Cover slip 3-5 min at room temp
Observe at 200-400X
Cells with high glycogen (notably vaginal and
penile urethral epithelial cells) will exhibit a
chocolate-brown color. Other epithelial cells
will exhibit a yellow color.

83. M L Importance
• Identification – DNA analysis of the endothelial
cells of Vaginal fluid & stains
• Sex determination – Barr body examination in
these endothelial cells
• Vaginal fluid & stain on a particular place –
connect the person with the scene of crime
• Identification of accused/victim in cases of
sexual assaults (Rape) – stains/endothelial cells
on the external genatelia of each others

84. Hair and Fiber
• Sampling
• Handling
• Packing
• Storage
• Examination

85. • Handling, Packing & Storage
• Photograph the item prior to conducting
any analyses.
• Document and remove other evidence
(e.g. fiber, blood, semen, paint etc) that
may require additional analysis.
• Document and record descriptions of any
physical damage (e.g., cut, crushed,
singed etc).
• Dimensions: size, length, diameter etc.

86. Temporary Mount:
• Make a temporary mount of the hair
sample on a clean slide with the distilled
H2O or glycerin. Cover with cover slip.

87. Scale Casting:
Nail Polish/Cellulose Acetate Method:
• Cleaned before examination with suitable
detergent
Hair Nail polish
• Paste and press with another clean slide
• Dry for 2-5 min
• Fine forceps, lift the hair from root end
• Observe the scale impressions by
microscope

88. Human Hair Scales:

89. Permanent Mount:
• Place hair on slide in a drop of xylene
and add permanent mounting medium.
• Place a cover slip on the hair allowing
the medium to spread under cover slip-
encasing hair.
• Label the slide appropriately and allow it
to dry for 48 hours.

90. Cross Sectioning:
• Clean hair in ether: ethanol (1:1) mixture.
• Bundle the samples and dip in a block of
molten wax and allow to cool. Keep in
refrigerator for 2-3 hours.
• Cross sections can be taken either with a
sharp blade or with a microtone to a thickness
of 5-10 microns.
• Place sections on a clean slide and dissolve
wax with a drop of xylene.
• Prepare permanent mount of the sections and
examine under the microscope.

92. • Examination:
• Hair/fiber
• If hair- human/animal
• If human- from which part of body
- child/adult/old age
- natural/dye is used (color)
• Sex determination from hair root sheath
• Blood grouping from hair root

93. Comparison
Microscope

94. Feature Human Hair Animal Hair
Color Relatively consistent
along shaft
Often showing profound
color changes and banding
Cortex Occupying most of the
width of shaft greater
than medulla
Usually less than width of
medulla
Distributio
n of
pigment
Even, slightly more
towards cuticle
Central or denser towards
medulla
Medulla Less than one-third width
of shaft. Amorphous,
mostly not continuous
when present
Greater than one-third width
of shaft. Continuous, often
varying in appearance along
shaft, defined structure
Scales Imbricate, similar along
shaft from root to tip
Often showing variation in
structure along shaft from
root to tip

95. Race Diamete
r
Cross-
section
Pigmentati
on
Cuticle Undul
ation
Negroid 60 – 90
um
Flat Dense &
clumped
- Prevale
nt
Caucas
oid
70 – 100
um
Oval Evenly
distributed
Medium Uncom
mon
Mongol
oid
90 – 120
um
Round Dense Thick Never

96. • Scalp- Head hair, 100 – 1000 mm long,
25-125 um diameter, 0.4 mm/day growth;
small root, tapered tip, little diameter
variation, various medullation, often with
cut tips, may artificially treated.
• Pubic- Pudental, 10 – 60 mm long, coarse
and prominent diameter, variation and
buckling, broad medulla, follicular tags
common, asymmetrical cross section
twisted and constricted, may be straight,
curved or spirally tufted.

97. • Vulvar- Secondary pubic hair, finer and
shorter than pubic.
• Chest- Pectoral, moderate to
considerable diameter variation, long fine
arch-like tip, usually longer than pubic.
• Beard- Facial hair, very coarse, 50-300
mm long, large root, irregular structure,
often triangular cross section, complex
medullation, blunted or razor cut tips.

98. • Axillary- Arm pit, 10-50 mm long, grows
0.3 mm/day, coarse, blunt tip, abraided or
frayed, usually straighter than pubic hair,
many cortical fusi, sometimes yellowed
and bleached.
• Eyebrow- Superciliary, 1 cm long, curved,
relatively coarse for length, smooth curve
with punctate tip and large medulla.
• Eyelash- Ciliary, less than 1 cm long,
short curved pointed hair.

99. • Limb- Leg and arm hair, 3-6 mm long, fine
tips, irregularly medullated, often
indistinctly, slightly pigmented.
• Ear- Tragi, pinnae, down.
• Buttocks- Anal hair, short blunted and
abraided hair.
• Nose- Similar to facial hair.

100. • Sex determination from hair root sheath
+ drop of MgCl2
• Cover with Quinacrine dihydrochloride soln
• Wash with MgCl2 soln
add a drop of glycerol
solution and cover slip.
• Y-body under UV light in Fluorescent
Microscope
• Males show >25% of nuclei +ve

101. • Blood grouping from hair root
- If fresh blood is present by slide method
- Stains – absorption elution technique

102. M L Importance
• Identification – blood grouping & sex
determination (Barr body) by hair root
sheath
• Cause of death – heavy metal poisons
(As, Cu etc) can be detected even after
decades

103. • Crimes
- Stains (blood, semen, vomit etc) on hairs
will connect the victim/accused to each
other & to the nature of assault
- Cut/crushed hairs – type of weapon used
- Connect the person with crime scene

104. - Singeing of hair – burns & also
differentiate from scalds
- In rape & sodomy pubic hairs may be
found on accused/victim of each others
• Age of a person by graying of hair
(child/adult/old age)

105. Fiber Examination:
• Sampling
• Handling
• Packing
• Storage
• Examination - Microscopical examination
- Staining tests
- Solubility tests
- Physical methods of analysis.

106. • Handling, Packing & Storage
• Photograph the item prior to conducting
any analyses.
• Document and remove other evidence
(e.g. hair, blood, semen, paint etc) that
may require additional analysis.
• Document and record descriptions of any
physical damage (e.g., worn, cut, broken
and frayed etc).

107. • Severed ends for possible physical
matches
• Knots, ligatures or both
• Dimensions: size, length, diameter etc.
• Components: number, type and twist.
• Color
• Dyes

108. • Do not bring a questioned specimen in
contact with the known fabric (e.g., piece
of fabric, yarn and tuft of fibers etc)
• Do not alter the condition of a questioned
specimen (e.g. shape, position, layers, or
relation of one yarn to another)
• Do not cut a sample to be used for
comparison testing from ends of yarn or
edges of fabric if there is a possibility of
physically matching a questioned
specimen

109. Microscopic Examination:
Temporary Mount:
• Place small quantity of fiber on a slide.
• Add a drop of distilled water or glycerine and
place a cover slip.
• Examine under the microscope. A study of cell
wall and lumen, crystals, fiber ends,
dislocations and cross markings, cells from
tissues other than sclerenchyma will help in
the identification of plant fibers.

110. Cotton Wool Linen Nylon Silk Rayon

111. Microscope equipped for polarized
light & fluorescence examinations
for analyzing hair/fiber

112. M L Importance
• Crimes
- Cut/crushed fiber – type of weapon used
- Connect the person with crime scene

113. Trace evidence
collection kit

114. REFERENCES
• Vanezis P, Bustil A. Suspicious Death
Scene Investigation. Arnold, New York.
1996.
• Director of forensic science, MHA, Govt
of India. Laboratory procedure manual
FORENSIC BIOLOGY.
• Reddy K S N. The Essentials of Forensic
Medicine and Toxicology. Medical Book
Company, Hyderabad. 2007: 51-88, 388-
405.

115. • Houck M M. Trace Evidence Analysis.
Elsevier, London. 2004. 1-26.
• Sukko P, Knight B. Knights Forensic
Pathology. Arnold, London. 2004: 421-
429.
• Guharaj P V, Chandran M R. Forensic
Medicine. Orient Longman, Hyderabad.
2003: 262-287, 438-443.
• Mathiharan K, Amrit K P. Modis Medical
Jurisprudence and Toxicology.
LexisNexis, New Delhi. 2006: 471-559.