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LAST DATE - 14/04/23

7 Leishman staining and microscopic examination of blood

smear
Theory - Leishman Stain is a neutral stain for blood smears which was
devised by the British surgeon W. B. Leishman (1865–1926). It
consists of a mixture of eosin (an acidic stain), and Methylene blue (a
basic stain) in Methyl alcohol and is usually diluted and buffered
during the staining procedure. It stains the different components of
blood in a range of shades between red and blue.
It is based on a methanolic mixture of “polychromed” Methylene blue
and eosin. The methanolic stock solution is stable and also serves the
purpose of directly fixing the smear eliminating a prefixing step.
The working principle of the Leishman stain is same as described
above. As it is a type of Romanowsky stains, it contains both the Acidic
and Basic dyes which have the affinity for Basic and Acidic
components of the Blood cells respectively. The acidic dye, Eosin,
variably stains the Basic components of the cells i.e. the cytoplasm,
Granules etc. and the Basic dye, Methylene blue stains the Acidic
components, especially the Nucleus of the cell. The stain must be
diluted for use with Phosphate buffer to pH 6.8 or 7.2, depending on
the specific technique used. The pH 6.8 is preferred when the
morphology of blood cells is to be examined and pH 7.2 is good for
parasitic studies.
One of the major forensic significance leishman staning holds is
gender determination for visualizing Barr bodies from cytosmears
of
buccal mucosa.

Materials required - refer to previous year TMFS PRAC.

Procedure - refer to previous year TMFS PRAC
Results -refer to previous year TMFS PRAC
precautions-refer to previous year TMFS PRAC
8 Documentation of blood splatter

● Shared pdf . (exclude abstract, read carefully if you find any

matter irrelevant skip)
● Theory - Include introduction,serological
analysis,photography & sketches given in the pdf.

Materials required - Various size scales

Magnifier Loop with scale
Tape measure
Level
Colored string
Tape
Procedure- All items of evidence shall be marked with the
proper case number, item number, and other identifying
marks when possible.
● A visual exam shall be performed in which the FS/CSI
attempts to identify any bloodstain pattern(s) present
and classify them based on physical characteristics.
Once the pattern(s) are classified, a bloodstain pattern
should be evaluated for the presence of any additional
information the stain(s) may exhibit. All observations
shall be recorded in the case notes. If the FS/CSI is not
performing their own serological examinations, they
should wait to issue the BPA report until after the
Certificate of Analysis for the associated serology
testing has been issued.
● If the FS/CSI is performing their own serological
examination of the evidence as well as the pattern
 analysis, the FS/CSI shall be proficient in the tests
performed.
● If serological testing will alter the stain or pattern
rendering it unsuitable for further analysis, thorough
documentation and pattern analysis shall be completed
prior to serological testing.
● If it is necessary to remove an entire stain, it shall be
documented in notes and photographed.
● Notes and sketches shall document the item(s) of
evidence and the observed bloodstains including the
following: number, locations, sizes, shapes,
directionality and any additional observations (condition
of blood: dry, color, etc.). Sketch templates are
available on the network drive.
● precautions- shall wear appropriate personal protective
equipment .
Result- the given documentation of the given blood spatter
pattern was successfully done.

9 Interpretation of blood patterns - PART A

Theory- Bloodstains range in both amount of blood and type of pattern—from pools of blood
around a body to obvious spatter patterns on the walls to microscopic drops on a suspect’s clothing.
The shape of the bloodstain pattern will depend greatly on the force used to propel the blood as
well as the surface it lands on. Forward spatter from a gunshot wound will typically form smaller
droplets spread over a wide area, while impact spatter will form larger drops and be more
concentrated in the areas directly adjacent to the action.
Because blood demonstrates surface tension, or cohesive forces that act like an outer skin, a drop of
blood dropped at a 90° angle forms a near-perfect spherical shape. A smooth surface, such as tile or
linoleum, will cause little distortion of this spherical shaped drop, whereas a rougher surface, such as
carpet or concrete, disrupts the surface tension and causes the drops to break apart.
The number and location of stains, as well as the volume of blood influence how much useful
information can be gathered. Large amounts of blood, such as if the person bled to death or was so
severely injured that the resulting blood spatter was extensive, can often yield less information than
several well-defined spatter patterns. Too much blood can disguise spatter or make stain patterns
unrecognizable. Conversely, too little blood, just one or two drops, will likely yield little or no usable
information.

Stains that overlap or come from multiple sources present challenges to analysts, but often reveal
valuable details about the crime. Overlapping stains may obscure pattern details, but can provide
information on the force, timing and instrument used. In the case of multiple victims, analysts will
often use DNA profiling to determine whose blood is included in a given pattern, helping to
estimate the locations of the victims in relation to each other and the perpetrator(s)

Procedure -

● Blood was dropped from different height

● A different amount of blood was dropped from the same height onto a sheet of paper.
● Blood was dropped from a consistent height onto both porous and non- porous surfaces

Observation -

It should be in the form of table below the photograph or sheet of paper you are pasting

Total no. of observation table should be equal to no. of steps in procedure.

Result

Precaution

Note

Total no. of sheets for Part (A) - 3

1. Blood spatter from same height different volume(given)

2. Blood spatter from different height (given)
3. Graph sheet (given)
PART-B

Determination Impact angle, point of origin & height of blood spatter

theory-Blood spatter analysis helps crime-scene investigators reconstruct what happened at
the crime scene. Using only blood spatter analysis, you may be able to recognize the events
leading up to the crime. Although crime scene investigators may arrive at the crime scene after
the victim and witnesses are no longer present, they still need to determine what happened.
Often several witnesses give different accounts of the crime. Which witness is providing an
accurate description of what really happened? During the investigation, the crime-scene
investigators need to determine if the evidence, in this case the blood spatter, matches the
description given by the witnesses, the suspect(s), and the victim(s). In domestic abuse cases,
the victim of domestic abuse may tell a false story to try to protect the abusing partner. A victim
may state that a head injury occurred as a result of falling down the stairs. However, if the blood
spatter patterns are inconsistent with this type of injury, then what type of injury did cause the
blood spatter? What actually happened? Is the witness lying? Further investigation is required
when the blood spatter evidence tells a different story than the witness’s account of the incident.
In this lab, you will analyze blood spatter in three dimensions. By noting the shape of the droplet
of blood, you will be able to note the direction in which the blood was moving. The size of the
blood spatter will provide some indication of the velocity of the blood when it hit the surface. By
examining at least two drops of blood spatter, you will be able to determine where the injured
person was located when the injury occurred in two dimensions (line so convergence). You can
easily measure the distance from the area of convergence to the drop of blood. If you want to
determine the point of origin, or height fro he impact surface, you will need to make some
calculations. By measuring the width and length of a single drop of blood, you can determine the
angle of impact. By using the Law of Tangents, you can calculate the height from which the
blood fell, or the point of origin for the blood.
Procedure - 1. Determine the direction of blood flow in the drops that follow with an arrow next
to the droplet.
a. If the blood drop is circular, then the blood fell at a 90o angle.
b. If it is not circular, then the angle of impact was less than 90o .
c. The elongated end of a drop of blood points to the direction in which the blood was moving.
2. From several drops of blood, determine the area of convergence by drawing lines through
each of the blood droplets and noting where the lines intersect.
3. Once you have determined the area of convergence, you will measure the distance from the
area of convergence to the edge of the drop of blood when it first impacted a surface. The
distance is indicated by the dotted line. Recall the diagram of a right triangle. The dotted line
represents the adjacent side of the angle of impact. 3 of 7
4. Next determine the angle of impact for each droplet of blood.
a. Select one of the blood droplets and determine the angle of impact for that drop of blood.
b. To calculate the angle of impact, you will need to use the Law of Sines.
c. Remember, when you measure the length of the blood droplet, do not include the thin
extension of the leading edge (tail). Sin of the angle of impact = width of the blood / length of the
blood drop Sin of the angle = width / length = …………………….

result & precaution

Total no. of working sheets for Part-B

1. Impact angle- working sheet (given)
2. Area of convergence- Diagram
3. Height of person - Diagram
10.Examination of seminal stains

Theory-
Semen is a human 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 3 ml per ejaculate.

No. of Spermatozoa= 50,000 to 3,50,000 per ml. (10% of SP).
It is a suspension of spermatozoa in seminal plasma.
Semen Contains the Following
1. Spermatozoa (10%)

2. Seminal plasma (90%)

3.Epithelial cell (<1%)

The spermatozoa are produced in the testis by the process of

spermatogenesis. Spermatozoa have within lipid proteins like protamine
and histone and so on and enzymes like dehydrogenases and
Transaminases. The total length of spermatozoa is about 50 it from of head
and tail. The head is flat, oval formed 4.6×2.6×1.6 in length distance from
side to side thickness. The Nucleus is living in a major part of the head.
The tail part is responsible for the motion of sperm. The seminal plasma is
a mixture of secretion derived from the male accessory reproductive organs
like epididymis, seminal vesicles, the prostate, vasa- deferential,
Bulbourethral, and urethral glands. The seminal plasma has in it citric acid,
ascorbic acid, lactic acid, Fructose, potassium, choline phosphatase,
proteases, free amino acids, Ergothioneine zinc, calcium, spermine lipids,
enzymes like Fibrinogenase, distance, acid, and alkaline, phosphatase
glycosidases, A and b Glucosidases, mannoxidases B Givcouridases.

The seeing who the person is of semen is of great value in Medicolegal

practice like put for supposed rape cases or sexual assault. The primary
purpose of semen forensic analysis is to sample and look at marks and
other biological material taken from the attackers the sufferer or
color-damaged spots discovered on clothes and or other evidence at the
crime place. In cases of sexual assault 2 steps are needed: give the
position of the stain and prove its identity. A semen analysis can also help
in successfully connecting semen to a person likely to wrongdoing through
DNA making common with a group.

As the number of cases of Rape are increasing day by day there is a need
of detection of sperm and semen even if present in small quantity, no
matter how old the stains are, we have to detect these stains precisely. For
detection of sperm and semen are the most reliable marker for the
investigation in cases of rape, sodomy, Bestiality, sexual murder etc.
detection of sperm is an important factor in confirming sexual assaults. This
paper provides a brief review and knowhow of the need for detection of
sperms and semen and various methods applied for the detection with
special emphasis on the current chemical tests with accurate procedures
for performing these tests.
The detection of semen in forensic science is essential in cases of sexual
assault but can be problematic in the absence of spermatozoa. Choline is
known to occur in high concentrations in seminal fluid and the Florence
Iodine test for its detection has been used in forensic science for many
years.
On the other hand The Barberio test relies on the formation of spermine
phosphate or picrate crystals upon reaction of an appropriate extract from
the suspected stain with appropriate anions.

Materials required
Chemicals required- Florence & Barberio’s reagent
Procedure-
1. Detection of choline - Florence test
A few drops of watery solution of the stain is extracted and taken on a slide
and a drop of Florence reagent (8%) W/V solution of Iodine in water
containing 5% W/V of Potassium Iodide) is poured & allowed to mix slowly
under a cover slip. Dark brown crystals of choline periodide, generally
needle shaped, formed with a few minutes. Non-specifics & false negative
results are common.

2.Detection of Spermine - Barberio’s test

Procedure: A few drops of Berberio’s reagent when added to spermatic
fluid produces crystals of sperm in picrate (needle shaped, rhombic & of
yellow color).
For various valid reasons, like non-specificity and lack of reproducibility, the
florence and berberio’s tests have not been accepted universally.

Observation -

Result -

Precaution -