ESTIMATION OF AGE OF DRIED BLOOD SATIN

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TITLEESTIMATION OF AGE OF DRIED BLOOD STAIN
BYUV-VIS SPECTROSCOPY

• BY
• NIHA ANSARI

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DECLARATION

• The work done here belongs to NIHA ANSARI only
  and any person found to be copying the work or
  presenting it anywhere will be considered as
  unlawful and that person will have to face legal
  actions.

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INTRODUCTION-

• Blood can be described as an specialized
  connective tissue in which there is liquid
  intercellular substances known as plasma and
  formed elements Red blood cells, white blood
  cells and platelets suspended in plasma and it
  also have proteins
• Blood contains Haemoglobin which is present in
  Red blood cells. Haemoglobin contains 96 of
  protein globin and 4 haem.
• In adult humans, the most common haemoglobin type
  is a tetramar called hemoglobin A, consisting of
  two a and two ß subunits non-covalently bound.

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• 
  Structure of Haemoglobin
• Changes in color of blood also occur from red to
  reddish brown to even dark brown or black.
• After death, gradually blood pH falls due to
  glycogenolysis, glycolysis, terminal accumulation
  of carbon dioxide, lactic acid, phosphoric acid
  and splitting of amino acids and fatty acids.

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AIM OF THE STUDY-

• Blood contain haemoglobin which has 96 of
  protein. These proteins undergo denaturation
  which may be due to shaking, change in
  temperature, change in reaction, addition of
  neutral salts etc.
• So this property of proteins that is denaturation
  which occurs with time can help us to correlate
  between age of the dried blood stain and protein
  denaturation with Temperature and Humidity.
• We can also correlate age of the dried blood
  stain and protein denaturation with age
  difference in Females.

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• SO,
• My first aim is to study the changes in
  the absorbance using UltraVioletvisible
  spectroscopy, which occurs in the dried blood
  stain due to the protein denaturation with time ,
  in relation to the temperature and humidity.
• My second aim is to study these changes
  in relation to the difference in Adult and
  Sub-adult female .

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METHODOLOGY-

• A. SAMPLE COLLECTION-
• Blood samples were collected from Case-1 and
  Case-2 from vein by percutaneous puncture using 5
  ml syringe. Then spots were placed on two white
  cotton cloth piece.

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• B. SAMPLE PREPARATION-
• For sample preparation samples were collected in
  the duration of seven days from both indoor and
  outdoor pieces of cloth.
• Samples were collected at following time duration
  on First day in case of both indoor and outdoor
  samples-
• 15 min, 30 min, 60 min, 90 min, 120 min, 3
  hr and 6 hr.
• Than samples were collected after 24 hr, 3rd,
  4th, 6th and 7th day in case of both indoor and
  outdoor.
• For each sample preparation 1cm1cm piece of
  cloth was taken then that cut piece of cloth was
  then dissolve in Tris HCL and then the samples
  were incubated for overnight.

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• Then on next day blood extract was taken into it
  Tris HCL was added and centrifuged.
• Supernatant was collected and the same was used
  for analysis in the UV-VIS spectroscopy.

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RESULT AND DISCUSSION-

• Results of Case-1 -
• The fresh samples of blood showed peaks at 540 nm
  which may be of ß-oxyhaemoglobin having
  absorbance 0.06.
• In indoor samples of first day of time duration
  of 15 min, 30 min, 60 min, 90 min, 120 min, 3 hr
  and 6 hr, the wavelength showed decrease from 413
  nm to 412 nm and the absorbance was from 0.78 to
  0.63.
• And in outdoor samples the wavelength showed
  decrease from 412 nm to 411 nm and there were
  increases in absorbance from 0.62 to 0.88.

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• The following is the graphical representation of
  the absorbance obtained during the time duration
  15 min, 30 min, 60 min, 90 min, 120 min, 180 min,
  6 hr, 24 hr, 3rd, 4th, 6th and 7th day.

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Results of Case-2-

• The fresh samples of blood showed peaks at 550 nm
  which may be of ß-oxyhaemoglobin having
  absorbance 0.25.
• In indoor samples of first day of time duration
  of 15 min, 30 min, 60 min, 90 min, 120 min, 3 hr
  and 6 hr, the wavelength remain 410 nm throughout
  and the absorbance vary from 1.0 to 1.09.
• And in outdoor samples collected the wavelength
  showed decrease from 410 to 409 and absorbance
  decreases from 1.2 to 0.86.

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• The following is the graphical representation of
  the absorbance's obtains during the time
  duration 15 min, 30 min, 60 min, 90 min, 120
  min, 180 min, 6 hr, 24 hr, 3rd, 4th, 6th and 7th
  day.

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DISCUSSION-

• As we know haemoglobin has 96 protein in it.
  These proteins present in it do undergo many
  changes chemically and physically both. 
• Denaturation of proteins involves the disruption
  and possible destruction of both the secondary
  and tertiary structures. Denaturation disrupts
  the normal alpha-helix and beta sheets in a
  protein and uncoils it into a random shape.
• Heat can disrupt hydrogen bonds and non-polar
  hydrophobic interactions in protein. 

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• Due to this denaturation of protein haemoglobin
  get converted into its various form and these
  results in changes in the absorbance of the blood
  extract samples.
• In our body haemoglobin is mixed with oxygen that
  is oxyhaemoglobin in which iron is in ferrous
  state. Then irons get converted to ferric state
  thus forming methaemoglobin.

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• We also see formation of haemochromogen as it is
  a haem with ferrous iron combined with denatured
  globin. Later cathaemoglobin a compound of haem
  containing ferric iron with denatured globin may
  also have been formed.

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CONCLUSION-

• From the results obtain it can be concluded that
  to some extend the technique UV-VIS spectroscopy
  can be use to determine the age of the dried
  blood stain.
• As samples were taken from adult (Case-2) and
  sub-adult (Case-1) female the result obtain show
  changes in absorbance and wavelength. There are
  not any significant changes in the results of
  adult and sub-adult female in both wavelength
  show decrease and absorbance also.
• It can be said that as time increases both
  wavelength and absorbance decreases.

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• In Case-1, the wavelength of outdoor samples
  decreases from 412 nm to 409 nm and in indoor
  samples it decreases from 413 nm to 409 nm.
• The absorbance decreases from 0.78 to 0.58 in
  indoor samples and in outdoor samples absorbance
  decreases from 0.62 to 0.12 in duration of seven
  days.
• In Case-2, the wavelength of indoor and outdoor
  samples varies between 410 nm 405 nm. The
  absorbance show decreases from 1.17 to 0.04 in
  indoor samples and in outdoor samples it shows
  increase from 1.0 to 2.0.
• We can also study further these changes by
  studying the protein denaturation further in
  detail.

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FORENSIC SIGNIFICANCE

• In most of the crime cases
  we find blood , so if we are able to estimate the
  age of a dried blood stain obtain from crime
  scene then we can be know many things which can
  help us in our investigation
• We can find out that how old the stain is?
• We can determine Time since Death if we are able
  to obtain accurate results.
• As in many cases we dont
  find any evidences or eye witnesses which can
  help us. We sometime find only dried biological
  stains present on the crime scene. So if we can
  know the age of dried blood stain we can
  determine the Time since Death.

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• If we find any death body at any place then from
  the dried blood stain we can estimate age of
  blood and can know how many days before the crime
  has been taken place.
• To some extend we can even know whether the blood
  stain obtained was exposed to heat for a longtime
  or not as exposures to heat bring absorbance at
  lower level.