FORENSIC‌‌MEDICINE‌‌AND‌‌TOXICOLOGY‌ 

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UNIT‌‌4:‌‌BODY‌‌FLUIDS‌‌AND‌‌DNA‌  ‌
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In‌‌the‌‌investigation‌‌of‌‌crimes‌‌such‌‌as‌‌Murder,‌‌robbery,‌‌rape,‌‌The‌‌examination‌‌of‌‌ 
biological‌‌materials‌‌play‌‌an‌‌important‌‌role‌‌in‌‌connecting‌‌the‌‌criminal‌‌with‌‌a‌‌crime.‌‌Such‌‌ 
biological‌‌specimens‌‌may‌‌be‌‌in‌‌the‌‌form‌‌of‌‌body‌‌Fluids‌‌stains‌‌or‌‌other‌‌materials‌‌like‌‌ 
blood,‌‌semen,‌‌saliva,‌‌urine‌‌Fecal‌‌matte,‌‌and‌‌milk.‌‌These‌‌are‌‌often‌‌the‌‌key‌‌in‌‌a‌‌criminal‌‌ 
investigation‌‌and‌‌are‌‌subsequently‌‌relied‌‌upon‌‌in‌‌court.‌‌Many‌‌body‌‌fluid‌‌stains‌‌are‌‌ 
invisible‌‌and‌‌present‌‌in‌‌very‌‌small‌‌quantities‌‌or‌‌mixtures,‌‌so‌‌identification‌‌is‌‌not‌‌always‌‌ 
straightforward.‌  ‌
 ‌
A. BLOOD‌  ‌
Blood‌‌is‌a‌ ‌‌body‌‌fluid‌‌in‌‌humans‌‌‌and‌‌other‌‌animals‌‌that‌‌delivers‌‌necessary‌‌substances‌‌ 
such‌‌as‌‌nutrients‌‌and‌‌oxygen‌‌to‌‌the‌‌cells‌‌and‌‌transports‌‌metabolic‌‌waste‌‌products‌‌away‌‌ 
from‌‌those‌‌same‌‌cells.‌  ‌
In‌‌humans,‌‌blood‌‌is‌‌an‌o‌ paque‌‌‌red‌‌fluid,‌‌freely‌‌flowing‌‌but‌‌denser‌‌and‌‌more‌‌viscous‌‌than‌‌ 
water.‌‌The‌‌characteristic‌‌color‌‌is‌‌imparted‌‌by‌h‌ emoglobin‌,‌‌a‌‌unique‌‌iron-containing‌‌ 
protein.‌‌Hemoglobin‌‌brightens‌‌in‌‌color‌‌when‌‌saturated‌‌with‌‌oxygen‌‌(oxyhemoglobin)‌‌ 
and‌‌darkens‌‌when‌‌oxygen‌‌is‌‌removed‌‌(deoxyhemoglobin).‌  ‌

Blood‌‌is‌‌conducted‌‌through‌‌blood‌‌vessels‌‌(arteries‌‌and‌‌veins).‌‌Blood‌‌is‌‌prevented‌‌from‌‌ 
clotting‌‌in‌‌the‌‌blood‌‌vessels‌‌by‌‌their‌‌smoothness,‌‌and‌‌the‌‌finely‌‌tuned‌‌balance‌‌of‌‌clotting‌‌ 
factors.‌  ‌

Types‌‌of‌‌Blood‌‌Cells‌  ‌
We‌‌
  have‌‌ seen‌‌ blood‌‌ consist‌‌ of‌‌ cells‌‌ known‌‌ as‌‌ formed‌‌ elements‌‌ of‌‌ blood.‌‌ These‌‌ cells‌‌ have‌‌ 
their‌  ‌own‌  ‌functions‌  ‌and‌  ‌roles‌  ‌to‌  ‌play‌  ‌in‌  ‌the‌  ‌body.‌  ‌The‌  ‌blood‌  ‌cells‌  ‌which‌  ‌circulate‌  ‌all‌‌ 
around‌‌the‌‌body‌‌are‌‌as‌‌follows:‌  ‌

Red‌‌blood‌‌cells‌‌(Erythrocytes)‌  ‌
RBCs‌  ‌are‌  ‌biconcave‌  ‌cells‌  ‌and‌  ‌without‌  ‌nucleus‌  ‌in‌  ‌humans,‌  ‌also‌  ‌known‌  ‌as‌  ‌erythrocytes.‌‌ 
RBCs‌  ‌contain‌  ‌the‌  ‌iron-rich‌  ‌protein‌  ‌called‌  ‌hemoglobin;‌  ‌gives‌  ‌blood‌  ‌its‌  ‌red‌  ‌color.‌  ‌RBCs‌‌ 
are‌‌  the‌‌
  most‌‌
  copious‌‌
  blood‌‌
  cells‌‌
  produced‌‌
  in‌‌
  the‌‌
  bone‌‌
  marrow.‌‌ Their‌‌ primary‌‌ function‌‌ is‌‌ 
to‌‌transport‌‌oxygen‌‌from‌‌and‌‌to‌‌various‌t‌issues‌‌‌and‌‌organs.‌  ‌

White‌‌blood‌‌cells‌‌(Leucocytes)‌  ‌
Leucocytes‌  ‌are‌  ‌colorless‌  ‌blood‌  ‌cells.‌  ‌They‌  ‌are‌  ‌colorless‌  ‌because‌  ‌it‌  ‌is‌  ‌devoid‌  ‌of‌‌ 
hemoglobin.‌‌   They‌‌ are‌‌ further‌‌ classified‌‌ as‌‌ granulocytes‌‌ and‌‌ agranulocytes.‌‌ WBCs‌‌ mainly‌‌ 
contribute‌‌to‌‌immunity‌‌and‌‌defense‌‌mechanism.‌  ‌

Functions‌‌of‌‌Blood‌‌‌[additional‌‌info]‌  ‌
Blood‌‌is‌‌responsible‌‌for‌‌the‌‌following‌‌body‌‌functions:‌  ‌

Fluid‌‌Connective‌‌Tissue:‌B ‌ lood‌‌is‌‌a‌‌fluid‌‌connective‌‌tissue‌‌composed‌‌of‌‌55%‌‌plasma,‌ 
and‌‌45%‌‌formed‌‌elements,‌‌including‌‌WBCs,‌‌RBCs,‌‌and‌‌platelets.‌‌Since‌‌these‌‌living‌‌cells‌‌ 
are‌‌suspended‌‌in‌‌plasma,‌‌blood‌‌is‌‌known‌‌as‌‌a‌‌fluid‌‌connective‌‌tissue‌‌and‌‌not‌‌just‌‌fluid.‌  ‌

Provides‌‌oxygen‌‌to‌‌the‌‌cells:‌B ‌ lood‌‌absorbs‌‌oxygen‌‌from‌‌the‌‌lungs‌‌and‌‌transports‌‌it‌‌to‌‌ 
different‌‌cells‌‌of‌‌the‌‌body.‌‌The‌‌waste‌‌carbon‌‌dioxide‌‌moves‌‌from‌‌the‌‌blood‌‌to‌‌the‌‌lungs‌‌ 
and‌‌is‌‌exhaled.‌  ‌

Transports‌‌Hormone‌‌and‌‌Nutrients:‌T ‌ he‌‌digested‌‌nutrients‌‌such‌‌as‌‌glucose,‌‌vitamins,‌‌ 
minerals,‌‌and‌‌proteins‌‌are‌‌absorbed‌‌into‌‌the‌‌blood‌‌through‌‌the‌‌capillaries‌‌in‌‌the‌‌villi‌‌ 
lining‌‌the‌‌small‌‌intestine.‌  ‌
The‌  ‌hormones‌  ‌secreted‌  ‌by‌  ‌the‌  ‌endocrine‌  ‌glands‌  ‌are‌  ‌also‌  ‌transported‌  ‌by‌  ‌the‌  ‌blood‌  ‌to‌‌ 
different‌‌organs‌‌and‌‌tissues.‌  ‌

Homeostasis:‌B ‌ lood‌‌helps‌‌to‌‌maintain‌‌the‌‌internal‌‌body‌‌temperature‌‌by‌‌absorbing‌‌or‌‌ 
releasing‌‌heat.‌  ‌

Blood‌‌Clotting‌‌at‌‌Site‌‌of‌‌Injury:‌T ‌ he‌‌platelets‌‌help‌‌in‌‌the‌‌clotting‌‌of‌‌blood‌‌at‌‌the‌‌site‌‌of‌‌ 
injury.‌‌Platelets‌‌along‌‌with‌‌the‌‌fibrin‌‌form‌‌clot‌‌at‌‌the‌‌wound‌‌site‌  ‌

Transport‌‌of‌‌waste‌‌to‌‌the‌‌Kidney‌‌and‌‌Liver:‌B ‌ lood‌‌enters‌‌the‌‌kidney‌‌where‌‌it‌‌is‌‌ 
filtered‌‌to‌‌remove‌‌nitrogenous‌‌waste‌‌out‌‌of‌‌the‌‌blood‌‌plasma.‌‌The‌‌toxins‌‌from‌‌the‌‌blood‌‌ 
are‌‌also‌‌removed‌‌by‌‌the‌‌liver.‌  ‌
 ‌

 ‌
Composition‌‌of‌‌Blood.‌ 

There‌‌   are‌‌
  many‌‌   cellular‌‌
  structures‌‌
  in‌‌
  the‌‌
  composition‌‌   of‌‌ blood.‌  ‌When‌‌ a ‌‌sample‌‌ of‌‌ blood‌‌ 
is‌  ‌spun‌  ‌in‌  ‌a ‌ ‌centrifuge‌  ‌machine,‌  ‌they‌  ‌separate‌  ‌into‌  ‌the‌  ‌following‌  ‌constituents:‌  ‌Plasma,‌‌ 
buffy‌‌coat,‌‌and‌‌erythrocytes.‌  ‌

Plasma‌  ‌
The‌‌   liquid‌‌  state‌‌
  of‌‌
  blood‌‌  can‌‌
  be‌‌
  contributed‌‌  to‌‌
  plasma‌‌   as‌‌
  it‌‌
  makes‌‌   up‌  ‌~55%‌‌ of‌‌ blood.‌‌ It‌‌ 
is‌  ‌pale‌  ‌yellow‌  ‌in‌  ‌color,‌  ‌and‌  ‌when‌  ‌separated,‌  ‌it‌  ‌consists‌  ‌of‌  ‌salts,‌  ‌nutrients,‌  ‌water,‌  ‌and‌‌ 
enzymes.‌‌   Blood‌‌   plasma‌‌  also‌‌ contains‌‌ important‌‌ proteins‌‌ and‌‌ other‌‌ components‌‌ necessary‌‌ 
for‌  ‌overall‌  ‌health.‌  ‌Hence,‌  ‌blood‌  ‌plasma‌  ‌transfusions‌  ‌are‌  ‌given‌  ‌to‌  ‌patients‌  ‌with‌  ‌liver‌‌ 
failure‌‌and‌‌life-threatening‌‌injuries.‌  ‌

Red‌‌Blood‌‌Cells‌‌(RBC)‌‌   ‌
Red‌  ‌blood‌  ‌cells‌  ‌consist‌  ‌of‌  ‌Haemoglobin,‌  ‌a ‌ ‌protein.‌  ‌They‌  ‌are‌  ‌produced‌  ‌by‌  ‌the‌  ‌bone‌‌ 
marrow‌‌to‌‌primarily‌‌carry‌‌oxygen‌‌to‌‌the‌‌body‌‌and‌‌carbon‌‌dioxide‌‌away‌‌from‌‌it.‌  ‌

White‌‌Blood‌‌Cells‌‌(WBC)‌  ‌
White‌  ‌blood‌  ‌cells‌  ‌are‌  ‌responsible‌  ‌for‌  ‌fighting‌  ‌foreign‌  ‌pathogens‌  ‌(such‌  ‌as‌  ‌bacteria,‌‌ 
viruses,‌  ‌fungi)‌  ‌that‌  ‌enter‌  ‌our‌  ‌bodies.‌  ‌They‌  ‌circulate‌  ‌throughout‌  ‌our‌  ‌body‌  ‌and‌  ‌originate‌‌ 
from‌‌the‌‌bone‌‌marrow.‌  ‌

Platelets‌‌[thrombocytes]‌  ‌
Tiny‌  ‌disc-shaped‌  ‌cells‌  ‌help‌  ‌regulate‌  ‌blood‌  ‌flow‌  ‌when‌  ‌any‌‌
  part‌‌
  of‌‌
  the‌‌
  body‌‌
  is‌‌
  damaged,‌‌ 
thereby‌‌aiding‌‌in‌‌fast‌‌recovery‌‌through‌‌clotting‌‌of‌‌blood.‌ 
The‌‌
  above-stated‌‌  elements‌‌ form‌‌ the‌‌ composition‌‌ of‌‌ blood‌‌ in‌‌ humans.‌‌ The‌‌ only‌‌ vertebrate‌‌ 
without‌  ‌hemoglobin‌  ‌is‌  ‌the‌  ‌crocodile‌  ‌icefish.‌  ‌It‌  ‌derives‌  ‌its‌  ‌oxygen‌  ‌requirement‌  ‌directly‌‌ 
from‌‌the‌‌cold,‌‌oxygen-rich‌‌water‌‌where‌‌it‌‌lives.‌  ‌

Blood‌‌Group‌‌System‌  ‌
Karl‌  ‌Landsteiner,‌  ‌an‌  ‌Austrian‌  ‌scientist,‌  ‌discovered‌  ‌the‌  ‌ABO‌  ‌blood‌  ‌group‌  ‌system‌  ‌in‌‌   the‌‌ 
year‌  ‌1900.‌  ‌In‌‌  his‌‌
  experiments,‌‌   he‌‌
  mixed‌‌  different‌‌   blood‌‌
  types.‌‌
  He‌‌   noted‌‌  that‌‌
  the‌‌
  plasma‌‌ 
from‌‌   certain‌‌  blood‌‌   types‌‌
  produced‌‌   agglutinates‌‌   or‌‌  formed‌‌  clusters‌‌ caused‌‌ by‌‌ the‌‌ absence‌‌ 
of‌  ‌molecules‌  ‌on‌  ‌red‌  ‌blood‌  ‌cells‌  ‌and‌  ‌resulting‌  ‌in‌  ‌antibodies‌  ‌to‌  ‌defeat‌  ‌that‌  ‌molecule.‌‌  He‌‌ 
then‌  ‌made‌  ‌a ‌ ‌note‌  ‌of‌  ‌the‌  ‌agglutination‌  ‌and‌  ‌divided‌  ‌the‌  ‌blood‌  ‌types‌  ‌into‌  ‌four‌  ‌different‌‌ 
groups.‌‌For‌‌the‌‌discovery‌‌of‌‌the‌‌ABO‌‌blood‌‌group,‌‌he‌‌was‌‌awarded‌‌the‌‌Nobel‌‌Prize.‌  ‌
The‌  ‌blood‌  ‌grouping‌  ‌system‌  ‌is‌  ‌pivotal‌  ‌in‌  ‌blood‌  ‌transfusion.‌  ‌Our‌  ‌immune‌  ‌system‌‌ 
recognizes‌‌   another‌‌
  blood‌‌  type‌‌  as‌‌
  foreign‌‌
  and‌‌  attacks‌‌
  it‌‌
  if‌‌
  introduced‌‌ in‌‌ the‌‌ body,‌‌ causing‌‌ 
a‌  ‌transfusion‌  ‌reaction‌. ‌ ‌Any‌  ‌inappropriate‌  ‌match‌  ‌with‌  ‌the‌  ‌Rh‌  ‌and‌  ‌ABO‌  ‌blood‌  ‌types‌‌ 
cause‌‌   the‌‌
  most‌‌
  serious‌‌
  and‌‌  life-threatening‌‌   transfusion‌‌ reactions.‌‌ Therefore,‌‌ before‌‌ blood‌‌ 
transfusion,‌‌it‌‌is‌‌suggested‌‌to‌‌have‌‌a‌‌blood‌‌group‌‌checked‌  ‌
During‌  ‌the‌  ‌blood‌  ‌transfusion,‌  ‌the‌  ‌two‌  ‌most‌  ‌important‌  ‌group‌  ‌systems‌  ‌examined‌  ‌are‌  ‌the‌‌ 
ABO‌‌system‌‌‌and‌‌the‌R ‌ hesus‌‌system‌. ‌
The‌‌  ABO‌‌  blood‌‌   group‌‌   system‌‌  consists‌‌
  of‌‌ 4 ‌‌types‌‌ of‌‌ blood‌‌ groups‌‌ – ‌‌A,‌‌ B,‌‌ AB,‌‌ and‌‌ O ‌‌and‌‌ 
is‌‌
  mainly‌‌
  based‌‌   on‌‌
  the‌‌
  antigens‌‌ and‌‌ antibodies‌‌ on‌‌ red‌‌ blood‌‌ cells‌‌ and‌‌ in‌‌ the‌‌ plasma.‌‌ Both‌‌ 
antigens‌  ‌and‌  ‌antibodies‌  ‌are‌  ‌protein‌  ‌molecules‌  ‌in‌  ‌which‌  ‌antigens‌  ‌are‌  ‌present‌  ‌on‌  ‌the‌‌ 
surface‌‌
  of‌‌
  Red‌‌  Blood‌‌   Cells‌‌
  and‌‌
  antibodies‌‌   are‌‌
  present‌‌   in‌‌ the‌‌ plasma‌‌ which‌‌ is‌‌ involved‌‌ in‌‌ 
defending‌‌mechanisms.‌  ‌
On‌  ‌the‌  ‌other‌  ‌hand,‌  ‌the‌  ‌Rh‌  ‌blood‌  ‌group‌  ‌system‌  ‌consists‌  ‌of‌  ‌50‌  ‌defined‌  ‌blood‌  ‌group‌‌ 
antigens.‌  ‌In‌‌
  the‌‌
  Rh‌‌
  system,‌‌   the‌‌
  most‌‌  important‌‌
  antigens‌‌
  are‌‌
  D,‌‌
  C,‌‌
  c,‌‌
  E,‌‌
  and‌‌
  e.‌‌
  The‌‌
  ABO‌‌ 
and‌‌Rh‌‌blood‌‌systems‌‌are‌‌discussed‌‌in‌‌detail‌‌below.‌  ‌

1.‌‌ABO‌‌blood‌‌group‌‌system‌  ‌
The‌  ‌basis‌  ‌of‌  ‌ABO‌  ‌grouping‌  ‌is‌  ‌of‌  ‌two‌  ‌antigens-‌  ‌Antigen‌  ‌A ‌ ‌and‌  ‌Antigen‌  ‌B.‌  ‌The‌  ‌ABO‌‌ 
grouping‌‌   system‌‌   is‌‌
  classified‌‌ into‌‌ four‌‌ types‌‌ based‌‌ on‌‌ the‌‌ presence‌‌ or‌‌ absence‌‌ of‌‌ antigens‌‌ 
on‌‌the‌‌red‌‌blood‌‌cells‌‌surface‌‌and‌‌plasma‌‌antibodies.‌  ‌
● Group‌‌A‌–‌ ‌‌contains‌‌antigen‌‌A‌‌and‌‌antibody‌‌B.‌  ‌
● Group‌‌B‌–‌ contains‌‌antigen‌‌B‌‌and‌‌antibody‌‌A.‌  ‌
● Group‌‌AB‌–‌ contains‌‌both‌‌A‌‌and‌‌B‌‌antigens‌‌and‌‌no‌‌antibodies‌‌(neither‌‌A‌‌nor‌‌B).‌  ‌
● Group‌‌O‌–‌ ‌‌contains‌‌neither‌‌A‌‌nor‌‌B‌‌antigen‌‌and‌‌both‌‌antibodies‌‌A‌‌and‌‌B.‌  ‌

2.‌‌Rh‌‌Blood‌‌Group‌‌System‌  ‌
In‌  ‌addition‌‌  to‌‌
  the‌‌  ABO‌‌   blood‌‌  grouping‌‌   system,‌‌   the‌‌
  other‌‌   prominent‌‌   one‌‌  is‌‌
  the‌‌
  Rh‌‌   blood‌‌ 
group‌  ‌system.‌  ‌About‌  ‌two-thirds‌  ‌of‌  ‌the‌  ‌population‌  ‌contains‌  ‌the‌  ‌third‌  ‌antigen‌  ‌on‌  ‌the‌‌ 
surface‌  ‌of‌‌
  their‌‌  red‌‌   blood‌‌  cells‌‌
  known‌‌   as‌‌
  ‌Rh‌‌  factor‌‌
  or‌‌  ‌Rh‌‌   antigen‌; ‌‌this‌‌
  decides‌‌   whether‌‌ 
the‌‌   blood‌‌
  group‌‌   is‌‌
  positive‌‌ or‌‌ negative.‌‌ If‌‌ the‌‌ Rh‌‌ factor‌‌ is‌‌ present,‌‌ an‌‌ individual‌‌ is‌‌ ‌rhesus‌‌ 
positive‌  ‌(Rh+ve);‌  ‌if‌‌   an‌‌
  Rh‌‌  factor‌‌
  is‌‌
  absent‌‌   individual‌‌   is‌‌  ‌rhesus‌‌   negative‌‌   ‌(Rh-ve)‌‌   as‌‌  they‌‌ 
produce‌‌Rh.‌  ‌
  ‌
 ‌
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GENERAL‌‌EXAMINATION‌‌OF‌‌BLOODSTAINS‌‌   ‌

(‌ 1)‌‌STAINS‌‌FOUND‌‌AT‌‌THE‌‌SCENE‌‌OF‌‌THE‌‌CRIME:‌‌One‌‌of‌‌the‌‌important‌‌ 
aspects‌‌of‌‌the‌‌visit‌‌to‌‌the‌‌scene‌‌of‌‌the‌‌crime‌‌is‌‌searching‌‌for‌‌and‌‌interpreting‌‌ 
bloodstains.‌‌Relatively‌‌minor‌‌blood‌‌smearing‌‌may‌‌also‌‌provide‌‌significant‌‌ 
evidence,‌‌such‌‌as‌‌a‌‌smear‌‌on‌‌the‌‌door‌‌handle.‌‌Heel‌‌prints‌‌or‌‌shoe‌‌prints‌‌on‌‌ 
bloodstained‌‌areas‌‌of‌‌the‌‌body,‌‌help‌‌in‌‌the‌‌identification‌‌of‌‌the‌‌assailant.‌‌The‌‌ 
distribution‌‌and‌‌amount‌‌of‌‌blood‌‌at‌‌the‌‌scene‌‌of‌‌the‌‌crime‌‌may‌‌give‌‌valuable‌‌ 
information‌‌about‌‌the‌‌manner‌‌of‌‌death,‌‌whether‌‌it‌‌was‌‌suicidal‌‌or‌‌to‌‌see‌‌clear‌‌drag‌‌ 
marks.‌‌Smears‌‌caused‌‌by‌‌fingers‌‌or‌‌palms‌‌are‌‌helpful‌‌in‌‌identification.‌‌A ‌‌
photograph‌‌of‌‌bloodstains‌‌at‌‌the‌‌scene‌‌of‌‌a‌‌crime‌‌is‌‌useful.‌‌   ‌

(2)‌‌PART‌‌OF‌‌THE‌‌BODY‌‌FROM‌‌WHICH‌‌STAIN‌‌IS‌‌DERIVED:‌ ‌MENSTRUAL‌‌ 
BLOOD‌‌is‌‌usually‌‌found‌‌on‌‌female‌‌garments,‌‌diapers,‌‌or‌‌pieces‌‌of‌‌cloth.‌‌It‌‌is‌‌dark‌‌ 
and‌‌fluid,‌‌has‌‌a‌‌disagreeable‌‌smell‌‌and‌‌the‌‌reaction‌‌is‌‌acid..•‌‌If‌‌the‌‌blood‌‌is‌‌from‌‌ 
the‌‌NOSE,‌‌mucus‌‌and‌‌hair‌‌from‌‌the‌‌nose‌‌may‌‌be‌‌found.‌‌VOMITED‌‌BLOOD‌‌is‌‌of‌‌ 
chocolate‌‌colour‌‌and‌‌acid‌‌in‌‌the‌‌reaction‌‌due‌‌to‌‌the‌‌action‌‌of‌‌gastric‌‌juice.‌‌Blood‌‌ 
due‌‌to‌‌HAEMOPTYSIS‌‌is‌‌bright‌‌red‌‌and‌‌frothy,‌‌with‌‌an‌‌alkaline‌‌reaction.‌‌blood‌‌ 
due‌‌to‌‌RAPE,‌‌semen,‌‌and‌‌pubic‌‌hair‌‌may‌‌be‌‌found.‌‌Bloodstains‌‌due‌‌to‌‌BOILS‌‌ 
AND‌‌SORES‌‌show‌‌a‌‌smeared‌‌appearance‌‌without‌‌definite‌‌drops‌‌of‌‌blood‌‌and‌‌ 
may‌‌contain‌‌pus‌‌cells‌‌and‌‌bacteria.‌  ‌

(‌ 3)‌‌AGE‌‌OF‌‌BLOOD‌‌STAINS:‌‌Fresh‌‌stains‌‌on‌‌light-colored‌‌clothes‌‌are‌‌of‌‌bright‌‌ 
red‌‌color,‌‌which‌‌gradually‌‌changes‌‌to‌‌reddish-brown‌‌in‌‌24‌‌hours,‌‌and‌‌brown‌‌ 
within‌‌a‌‌few‌‌days,‌‌which‌‌may‌‌become‌‌black‌‌after‌‌a‌‌long‌‌time.‌‌Fresh‌‌stains‌‌are‌‌ 
moist‌‌and‌‌sticky,‌‌and‌‌on‌‌drying,‌‌they‌‌stiffen‌‌the‌‌cloth‌‌because‌‌of‌‌the‌‌proteins.‌  ‌
‌On‌‌many‌‌metallic‌‌articles,‌‌bloodstains‌‌appear‌‌as‌‌dark‌‌shining‌‌spots‌‌or‌‌smears,‌‌and‌‌ 
when‌‌dry,‌‌show‌‌fissures‌‌and‌‌cracks.‌  ‌
‌In‌‌ordinary‌‌conditions,‌‌a‌‌drop‌‌of‌‌blood‌‌dries‌‌in‌‌an‌‌hour‌‌or‌‌two.‌‌If‌‌blood‌‌is‌‌ 
collected‌‌in‌‌pools,‌‌it‌‌may‌‌take‌‌12‌‌to‌‌36‌‌hours‌‌to‌‌dry,‌‌depending‌‌upon‌‌the‌‌size‌‌and‌‌ 
depth‌‌of‌‌the‌‌pool‌‌formed.‌‌   ‌
The‌‌recently‌‌shed‌‌arterial‌‌blood‌‌is‌‌bright-red‌‌and‌‌venous‌‌blood‌‌dark-red.‌‌The‌‌ 
solubility‌‌of‌‌bloodstains‌‌in‌‌water‌‌and‌‌other‌‌liquids‌‌depends‌‌mainly‌‌on‌‌the‌‌age‌‌of‌‌ 
the‌‌stains‌‌and‌‌the‌‌type‌‌of‌‌material‌‌on‌‌which‌‌it‌‌is‌‌found.‌‌   ‌
The‌‌fresher‌‌the‌‌blood,‌‌the‌‌more‌‌easily‌‌it‌‌is‌‌dissolved.‌‌The‌‌solubility‌‌gradually‌‌ 
diminishes‌‌with‌‌age.‌‌The‌‌age‌‌can‌‌also‌‌be‌‌determined‌‌by‌‌the‌‌spread‌‌of‌‌the‌‌soluble‌‌ 
ingredients‌‌like‌‌chlorides‌‌into‌‌the‌‌surrounding‌‌material.‌‌   ‌
Fluorescence‌‌decreases‌‌as‌‌the‌‌stain‌‌become‌‌older‌‌due‌‌to‌‌the‌‌increasing‌‌amount‌‌of‌‌ 
haematin.‌‌It‌‌can‌‌only‌‌be‌‌stated‌‌that‌‌the‌‌stain‌‌is‌‌very‌‌fresh,‌‌recent,‌‌some‌‌weeks,‌‌ 
months,‌‌or‌‌very‌‌old.‌  ‌

(‌ 4)‌‌SEX‌‌AND‌‌AGE‌‌OF‌‌PERSON:‌‌Sex‌‌can‌‌be‌‌determined‌‌from‌‌the‌‌presence‌‌of‌‌ 
sex‌‌chromatin‌‌in‌‌the‌‌leucocytes‌‌if‌‌the‌‌cells‌‌can‌‌be‌‌identified.‌‌At‌‌birth,‌‌the‌‌blood‌‌ 
forms‌‌a‌‌thinner‌‌and‌‌softer‌‌coagulum.‌‌The‌‌presence‌‌of‌‌fetal‌‌hemoglobin‌‌indicates‌‌ 
that‌‌the‌‌blood‌‌is‌‌derived‌‌from‌‌a‌‌child.‌‌   ‌

(5)‌‌LIVING‌‌OR‌‌DEAD‌‌BODY:‌‌Blood‌‌that‌‌has‌‌effused‌‌during‌‌life‌‌can‌‌be‌‌removed‌‌ 
in‌‌scales‌‌on‌‌drying,‌‌due‌‌to‌‌the·‌‌presence‌‌of‌‌fibrin.‌‌Blood‌‌that‌‌has‌‌flowed‌‌after‌‌ 
death‌‌tends‌‌to‌‌break‌‌up‌‌into‌‌a‌‌powder‌‌on‌‌drying.‌  ‌

(‌‌6)‌‌SOURCE‌‌OF‌‌BLOOD:‌‌If‌‌the‌‌victim‌‌and‌‌assailant‌‌are‌‌of‌‌different‌‌blood‌‌ 
groups,‌‌it‌‌is‌‌helpful‌‌in‌‌establishing‌‌the‌‌identity.‌‌If‌‌the‌‌stains‌‌are‌‌on‌‌the‌‌inner‌‌side‌‌of‌‌ 
the‌‌garment,‌‌they‌‌usually‌‌belong‌‌to‌‌the‌‌victim,‌‌but‌‌if‌‌found‌‌outside‌‌they‌‌may‌‌ 
belong‌‌to‌‌the‌‌victim‌‌or‌‌accused.‌‌   ‌

 ‌
Presumptive‌‌Tests‌  ‌
● Also‌‌known‌‌as‌‌preliminary‌‌tests,‌‌screening‌‌tests,‌‌or‌‌field‌‌tests‌  ‌
● Establish‌‌the‌p‌ ossibility‌‌‌that‌‌a‌‌specific‌‌bodily‌‌fluid‌‌is‌‌present‌  ‌
● Do‌‌not‌‌conclusively‌‌prove‌‌the‌‌presence‌‌of‌‌a‌‌specific‌‌substance‌  ‌
● Pros:‌‌Narrows‌‌possibilities,‌‌can‌‌be‌‌used‌‌on‌‌larger‌‌areas,‌‌can‌‌locate‌‌possible‌‌ 
evidence‌‌not‌‌visible‌‌to‌‌the‌‌naked‌‌eye,‌‌relatively‌‌inexpensive,‌‌and‌‌quick‌‌to‌‌ 
perform‌  ‌
● Cons:‌‌Risk‌‌of‌‌false‌‌positives‌‌and‌‌may‌‌be‌‌overly‌‌sensitive‌  ‌
● Uses:‌‌Provide‌‌initial‌‌information‌‌to‌‌determine‌‌what‌‌test‌‌to‌‌perform‌‌next,‌‌used‌‌in‌‌ 
combination‌‌with‌‌confirmatory‌‌tests‌  ‌
1. Benzdinide‌‌test/TMB‌‌test‌‌[refer‌‌record]‌  ‌
2. )‌‌Phenolphthalein‌‌Test‌‌(Kastle-Meyer‌‌Test):‌‌To‌‌a‌‌solution‌‌extracted‌‌from‌‌ 
the‌‌stain‌‌with‌‌distilled‌‌water,‌‌add‌‌ten‌‌to‌‌twenty‌‌drops‌‌of‌‌phenolphthalein‌‌ 
reagent‌‌(phenolphthalein‌‌2g.‌‌+‌‌sodium‌‌hydroxide‌‌20g.‌‌+‌‌zinc+‌‌distilled‌‌ 
water‌‌100‌‌ml),‌‌and‌‌then‌‌a‌‌drop‌‌or‌‌two‌‌of‌‌10‌‌volumes‌‌hydrogen‌‌peroxide.‌‌If‌‌ 
blood‌‌is‌‌present,‌‌a‌‌pink‌‌or‌‌purple‌‌color‌‌develops‌‌immediately.‌‌The‌‌test‌‌is‌‌ 
more‌‌specific‌‌for‌‌blood‌‌than‌‌the‌‌benzidine‌‌test,‌‌but‌‌comparatively‌‌less‌‌ 
sensitive.‌‌Traces‌‌of‌‌copper‌‌give‌‌a‌‌positive‌‌reaction.‌‌The‌‌tests‌‌employing‌‌ 
guaiacum‌‌(deep‌‌blue)‌‌and‌‌leucomalachite‌‌green‌‌are‌‌rarely‌‌used‌‌in‌‌ 
medico-legal‌‌work.‌‌   ‌

Confirmatory‌‌Tests‌  ‌
● Conclusively‌‌identify‌‌the‌‌identity‌‌of‌‌a‌‌biological‌‌material‌  ‌
● Maybe‌‌one‌‌or‌‌a‌‌combination‌‌of‌‌procedures‌  ‌
● Pros:‌‌Conclusively‌‌identifies‌‌a‌‌substance,‌‌smaller‌‌risk‌‌of‌‌false‌‌positives‌  ‌
● Cons:‌‌Maybe‌‌more‌‌expensive,‌‌require‌‌additional‌‌equipment,‌‌and‌‌take‌‌longer‌  ‌
1. Teichman‌‌Test‌‌[refer‌‌record]‌  ‌
2. Takayama‌‌test‌‌[refer‌‌record]‌  ‌

Medicolegal‌‌Application‌‌of‌‌Blood‌‌Groups‌‌and‌‌blood‌‌examination:‌‌   ‌
 ‌
(1)‌‌Disputed‌‌Paternity:‌‌‌The‌‌question‌‌of‌‌disputed‌‌paternity‌‌arises‌‌in‌‌the‌‌Court‌‌in‌‌the‌‌ 
following‌‌conditions.‌‌(1)‌‌When‌‌a‌‌child‌‌is‌‌born‌‌in‌‌lawful‌‌marriage,‌‌but‌‌the‌‌husband‌‌denies‌‌ 
that‌‌he‌‌is‌‌the‌‌father‌‌of‌‌the‌‌child.‌‌(2)‌‌When‌‌a‌‌child‌‌is‌‌born‌‌out‌‌of‌‌lawful‌‌marriage,‌‌and‌‌the‌‌ 
mother‌‌accuses‌‌a‌‌certain‌‌man‌‌of‌‌being‌‌the‌‌father‌‌of‌‌the‌‌child,‌‌while‌‌the‌‌man‌‌denies‌‌the‌‌ 
accusations.‌‌(3)‌‌When‌‌a‌‌woman‌‌pretends‌‌pregnancy‌‌and‌‌delivery‌‌and‌‌obtains‌‌a‌‌child‌‌ 
claiming‌‌it‌‌as‌‌her‌‌own,‌‌in‌‌order‌‌to‌‌obtain‌‌a‌‌share‌‌in‌‌her‌‌husband's‌‌property.‌‌(4)‌‌In‌‌suits‌‌for‌‌ 
nullity‌‌of‌‌marriage.‌  ‌
(2)‌‌Disputed‌‌Maternity:‌‌‌When‌‌the‌‌same‌‌child‌‌is‌‌claimed‌‌by‌‌two‌‌women,‌‌or‌‌when‌‌two‌‌ 
children‌‌are‌‌interchanged‌‌either‌‌by‌‌accident‌‌or‌‌by‌‌design‌‌in‌‌the‌‌maternity‌‌home‌‌or‌‌ 
hospital,‌‌or‌‌supposititious‌‌child,‌‌and‌‌when‌‌a‌‌woman‌‌kidnapping‌‌a‌‌child‌‌and‌‌claiming‌‌to‌‌ 
be‌‌the‌‌mother‌‌of‌‌that‌‌child,‌‌blood‌‌grouping‌‌tests‌‌are‌‌helpful.‌  ‌
‌(3)‌‌Crimes:‌‌‌Bloodstains‌‌may‌‌be‌‌found‌‌on‌‌the‌‌clothing‌‌and‌‌person‌‌of‌‌the‌‌suspect‌‌and‌‌ 
crime‌‌scene.‌  ‌
(4)‌‌Stains‌‌due‌‌to‌‌Body‌‌Fluids‌:‌‌The‌‌blood‌‌group‌‌agglutinogens‌‌can‌‌be‌‌demonstrated‌‌in‌‌ 
stains‌‌on‌‌clothes‌‌due‌‌to‌‌semen,‌‌sweat,‌‌saliva,‌‌nasal‌‌secretion,‌‌urine,‌‌or‌‌feces‌‌in‌‌persons‌‌ 
who‌‌are‌‌"secretors".‌‌This‌‌may‌‌be‌‌corroborative‌‌evidence‌‌of‌‌the‌‌accused.‌‌   ‌
(5)‌‌Identity:‌‌‌The‌‌specificity‌‌of‌‌various‌‌blood‌‌group‌‌combinations‌‌is‌‌like‌‌that‌‌of‌‌the‌‌ 
fingerprints.‌‌When‌‌an‌‌individual‌‌has‌‌some‌‌rare‌‌blood‌‌group,‌‌he‌‌can‌‌be‌‌identified‌‌with‌‌ 
certainty.‌‌But‌‌when‌‌they‌‌are‌‌of‌‌common‌‌type,‌‌they‌‌are‌‌not‌‌of‌‌use.‌‌   ‌
(6)‌‌Cause‌‌of‌‌Death:‌‌‌In‌‌certain‌‌cases,‌‌cause‌‌of‌‌death‌‌can‌‌be‌‌established,‌‌e.g.,‌‌ 
incompatible‌‌blood‌‌transfusion.‌‌Poisons‌‌can‌‌be‌‌detected‌‌in‌‌the‌‌blood.‌   ‌ ‌

B.‌S‌ EMEN‌  ‌
S‌emen‌‌is‌‌a‌‌greyish-white‌‌bodily‌‌fluid‌‌that‌‌is‌‌secreted‌‌by‌‌the‌‌gonads‌‌of‌‌male‌‌animals.‌‌It‌‌ 
carries‌‌sperm‌‌or‌‌the‌‌spermatozoa‌‌and‌‌fructose‌‌and‌‌other‌‌enzymes‌‌that‌‌help‌‌the‌‌sperm‌‌to‌‌ 
survive‌‌to‌‌facilitate‌‌successful‌‌fertilization.‌  ‌
t‌he‌‌cellular‌‌elements‌‌consist‌‌of‌‌spermatozoa‌‌and‌‌epithelial‌‌cells‌‌formed‌‌by‌‌the‌‌testes‌‌ 
semen‌‌is‌‌a‌‌thick‌‌white‌‌a‌‌pleasant‌‌secretion‌‌having‌‌characteristic‌‌order‌‌known‌‌as‌‌seminal‌‌ 
odor.‌  ‌
 ‌
The‌‌semen‌‌travels‌‌through‌‌the‌‌ejaculatory‌‌ducts‌‌and‌‌mixes‌‌with‌‌fluids‌‌from‌‌the‌‌seminal‌‌ 
vesicles,‌‌the‌‌prostate,‌‌and‌‌the‌‌bulbourethral‌‌glands.‌  ‌
The‌‌seminal‌‌vesicles‌‌produce‌‌a‌‌viscous,‌‌fructose-rich‌‌fluid‌‌forming‌‌around‌‌65-70%‌‌of‌‌the‌‌ 
semen‌‌base.‌  ‌
The‌‌white‌‌color‌‌of‌‌the‌‌semen‌‌is‌‌due‌‌to‌‌secretion‌‌from‌‌the‌‌prostate‌‌glands‌‌containing‌‌ 
enzymes,‌‌citric‌‌acid,‌‌lipids,‌‌and‌‌acid‌‌phosphatase.‌‌This‌‌forms‌‌around‌‌25-30%‌‌of‌‌the‌‌ 
semen‌‌base.‌  ‌
At‌‌each‌‌ejaculation,‌‌around‌‌200-500‌‌million‌‌sperms‌‌are‌‌released‌‌by‌‌the‌‌testes.‌‌This‌‌forms‌‌ 
about‌‌2-5%‌‌of‌‌the‌‌semen‌‌composition.‌  ‌
Apart‌‌from‌‌these,‌‌the‌‌bulbourethral‌‌glands‌‌produce‌‌a‌‌clear‌‌secretion.‌‌This‌‌helps‌‌in‌‌the‌‌ 
mobility‌‌of‌‌the‌‌sperm‌‌cells‌‌in‌‌the‌‌vagina‌‌and‌‌cervix.‌  ‌
Examination‌‌of‌‌Seminal‌‌Stain‌  ‌
 ‌
The‌‌examination‌‌of‌‌seminal‌‌stain‌‌may‌‌be‌‌carried‌‌by‌‌4‌‌methods‌‌which‌‌are‌‌physical,‌‌ 
chemical,‌‌microscopic,‌‌and‌‌electrophoretic.‌‌the‌‌materials‌‌of‌‌examination‌‌are‌‌collected‌‌as‌‌ 
follows‌‌: ‌ ‌
1.‌‌the‌‌portion‌‌of‌‌the‌‌fabric‌‌wearing‌‌the‌‌stain‌‌is‌‌cut‌‌dried‌‌in‌‌the‌‌shade‌‌to‌‌prevent‌‌ 
putrefaction‌‌and‌‌carefully‌‌preserved.‌  ‌
2.‌‌If‌‌pubic‌‌hair‌‌is‌‌matted‌‌together,‌‌a‌‌portion‌‌is‌‌cut‌‌and‌‌kept‌‌together.‌  ‌
3.‌‌To‌‌demonstrate‌‌the‌‌presence‌‌of‌‌semen‌‌in‌‌the‌‌vagina‌‌swaps‌‌are‌‌taken‌‌on‌‌sterile‌‌gauze‌‌or‌‌ 
cloth‌‌and‌‌Smear‌‌prepared‌‌on‌‌sterile‌‌slides‌  ‌
4.‌‌Dried‌‌a‌‌drawing‌‌seminal‌‌fluid‌‌on‌‌the‌‌thigh‌‌and‌‌perineum‌‌is‌‌collected‌‌on‌‌a‌‌piece‌‌of‌‌ 
constant‌‌cloth‌‌and‌‌tried.‌  ‌
5.‌‌dry‌‌stain‌‌on‌‌smooth‌‌surfaces‌‌is‌‌gently‌‌script‌‌with‌‌the‌‌clean‌‌plant‌‌scalpel‌‌into‌‌a‌‌glass‌‌ 
container‌  ‌

GENERAL‌‌EXAMINATION‌  ‌

Garments‌‌Submitted‌‌for‌‌laboratory‌‌examination‌‌are‌‌often‌‌dirty‌‌and‌‌may‌‌contain‌‌a‌‌variety‌‌ 
of‌‌stains;‌‌reddish‌‌color‌‌is‌‌commonly‌‌composed‌‌of‌‌blood;‌‌yellowish‌‌color‌‌for‌‌vaginal‌‌ 
discharge‌‌or‌‌urine,‌‌greyish‌‌appearance‌‌may‌‌be‌‌due‌‌to‌‌the‌‌semen.‌‌A‌‌preliminary‌‌ 
examination‌‌using‌‌UV‌‌light‌‌can‌‌be‌‌made‌‌where‌‌the‌‌stains‌‌of‌‌urine‌‌and‌‌seminal‌‌fluid‌‌will‌‌ 
show‌‌a‌‌bright‌‌fluorescence.‌‌the‌‌fluorescent‌‌seminal‌‌stains‌‌is‌‌a‌‌bluish-white‌‌color‌‌and‌‌such‌‌ 
stains‌‌are‌‌selected‌‌for‌‌further‌‌examination‌  ‌

Presumptive‌‌Tests‌  ‌

1.‌‌Acid‌‌Phosphatase‌‌Test:‌A ‌ lso‌‌known‌‌as‌‌the‌‌Walker‌‌Test‌‌or‌‌Brentamine‌‌spot‌‌test.‌‌The‌‌ 
male‌‌prostate‌‌gland‌‌produces‌‌and‌‌secrets‌‌into‌‌semen‌‌a‌‌high‌‌amount‌‌of‌‌the‌‌enzyme‌‌acid‌‌ 
phosphatase‌‌(AP).‌‌In‌‌the‌‌presence‌‌of‌‌Alpha-Naphthyl‌‌acid‌‌phosphate‌‌and‌‌Brentamine‌‌ 
Fast‌‌Blue,‌‌AP‌‌will‌‌produce‌‌a‌‌dark‌‌purple‌‌color‌‌in‌‌less‌‌than‌‌a‌‌minute.‌  ‌
2.‌‌Alternative‌‌Light‌‌Sources:‌‌‌Under‌‌specialized‌‌lights,‌‌semen‌‌will‌‌fluoresce‌‌due‌‌to‌‌the‌‌ 
presence‌‌of‌‌molecules‌‌such‌‌as‌‌Flavin‌‌and‌‌Choline-conjugated‌‌proteins.‌‌This‌‌color‌‌will‌‌ 
vary‌‌from‌‌blue‌‌to‌‌yellow‌‌depending‌‌on‌‌the‌‌light‌‌equipment‌‌used.‌  ‌
3.‌‌Prostate-Specific‌‌Antigen‌:‌‌Test‌‌detects‌‌prostate-specific‌‌antigen‌‌(PSA).‌‌PSA‌‌is‌‌ 
produced‌‌in‌‌high‌‌amounts‌‌by‌‌the‌‌male‌‌prostate‌‌gland‌  ‌

 ‌
Confirmatory‌‌Tests‌  ‌

1.‌‌Christmas‌‌Tree‌‌Stain:‌P ‌ ositive‌‌visual‌‌identification‌‌of‌‌sperm‌‌cells‌‌using‌‌a‌‌stain.‌‌Two‌‌ 
main‌‌reagents‌‌are‌‌used‌‌consecutively‌‌to‌‌produce‌‌this‌‌distinctive‌‌stain:‌‌ 
Picroindigocarmine‌‌stains‌‌the‌‌neck‌‌and‌‌tail‌‌portions‌‌of‌‌the‌‌sperm‌‌in‌‌green‌‌and‌‌blue,‌‌while‌‌ 
the‌‌Nuclear‌‌Fast‌‌Red‌‌(AKA‌‌Kernechtrot)‌‌gives‌‌the‌‌sperm‌‌heads‌‌a‌‌read‌‌color‌‌and‌‌the‌‌tip‌‌ 
of‌‌the‌‌heads‌‌a‌‌pink‌‌color.‌ 
2.‌‌R
‌ SID‌‌Test‌‌for‌‌Semen:‌I‌ dentifies‌‌the‌‌presence‌‌of‌‌the‌‌seminal‌‌vesicle-specific‌‌antigen‌‌ 
or‌‌semonogelin.‌‌This‌‌antigen‌‌is‌‌unique‌‌to‌‌human‌‌semen;‌‌therefore,‌‌there‌‌is‌‌no‌‌ 
cross-reactivity‌‌with‌‌other‌‌bodily‌‌fluids‌‌in‌‌males‌‌and‌‌females‌‌or‌‌with‌‌semen‌‌from‌‌other‌‌ 
mammals.‌‌This‌‌test‌‌can‌‌also‌‌identify‌‌semen‌‌even‌‌if‌‌the‌‌stain‌‌was‌‌stored‌‌in‌‌less‌‌favorable‌‌ 
conditions.‌‌   ‌

DETERMINATION‌‌OF‌‌BLOOD‌‌GROUP‌‌USING‌‌SEMEN‌  ‌

When‌  ‌semen‌  ‌is‌  ‌positively‌  ‌identified‌  ‌in‌  ‌vaginal‌  ‌fluid‌  ‌or‌  ‌other‌  ‌samples,‌  ‌a ‌ ‌test‌  ‌can‌  ‌be‌‌ 
carried‌‌
  out‌‌
  for‌‌
  the‌‌
  presence‌‌ of‌‌ blood‌‌ group‌‌ substances‌‌ in‌‌ the‌‌ same‌‌ sample.‌‌ The‌‌ ‘secretor’‌‌ 
individuals‌  ‌(80%‌  ‌individuals‌  ‌are‌  ‌secretors)‌  ‌will‌  ‌secrete‌  ‌the‌  ‌blood‌  ‌group‌  ‌substances‌  ‌in‌‌ 
body‌‌fluids,‌‌including‌‌semen.‌  ‌
 ‌

Medicolegal‌‌Application‌‌   ‌
 ‌
Semen‌‌is‌‌the‌‌second‌‌most‌‌common‌‌form‌‌of‌‌the‌‌material‌‌tested‌‌for‌‌DNA.‌‌It‌‌is‌‌almost‌‌ 
certainly‌‌the‌‌primary‌‌evidence‌‌in‌‌most‌‌sexual‌‌crimes‌‌such‌‌as‌‌rape.‌‌It‌‌is,‌‌in‌‌fact,‌‌the‌‌sperm‌‌ 
heads‌‌that‌‌contain‌‌the‌‌DNA‌‌rather‌‌than‌‌seminal‌‌fluid.‌‌Therefore,‌‌before‌‌DNA‌‌analysis‌‌is‌‌ 
performed,‌‌it‌‌is‌‌advisable‌‌to‌‌ascertain‌‌first‌‌whether‌‌the‌‌material‌‌is‌‌actually‌‌semen‌‌and‌‌ 
does‌‌contain‌‌sperm.‌‌Testing‌‌aspheric‌‌semen‌‌would‌‌clearly‌‌be‌‌of‌‌little‌‌value‌‌since‌‌there‌‌ 
would‌‌be‌‌no‌‌sperm‌‌present.‌‌Semen-stained‌‌clothing‌‌should‌‌similarly‌‌be‌‌examined‌‌to‌‌ 
confirm‌‌the‌‌presence‌‌of‌‌sperm.‌  ‌
 ‌
Semen‌‌may‌‌be‌‌found‌‌in‌‌liquid‌‌form‌‌or‌‌as‌‌dried‌‌smears‌‌or‌‌stains.‌‌Or,‌‌it‌‌may‌‌be‌‌found‌‌in‌‌ 
the‌‌vagina,‌‌anus,‌‌or‌‌rectum.‌‌Fresh‌‌semen‌‌is‌‌a‌‌gel-like‌‌fluid,‌‌which‌‌liquefies‌‌on‌‌exposure‌‌ 
to‌‌the‌‌atmosphere.‌‌Normal‌‌ejaculation‌‌is‌‌about‌‌3.4‌‌ml,‌‌containing‌‌about‌‌thirty‌‌million‌‌ 
sperms.‌‌The‌‌dry‌‌weight‌‌is‌‌about‌‌seven‌‌percent‌‌of‌‌the‌‌liquid‌‌weight.‌‌The‌‌sperm‌‌has‌‌a ‌‌
definite‌‌morphological‌‌structure.‌‌Its‌‌identification‌‌in‌‌a‌‌stain‌‌establishes‌‌its‌‌seminal‌‌nature.‌‌ 
The‌‌shape‌‌and‌‌size‌‌of‌‌a‌‌human‌‌spermatozoon‌‌are‌‌characteristic.‌‌But‌‌the‌‌morphology‌‌ 
alone‌‌does‌‌not‌‌permit‌‌individualization.‌  ‌
The‌‌semen,‌‌of‌‌some‌‌persons,‌‌does‌‌not‌‌contain‌‌a‌‌spermatozoon.‌‌It‌‌is‌‌called‌‌aspermic‌‌ 
semen.‌‌This‌‌may‌‌be‌‌due‌‌to‌‌some‌‌disease‌‌or‌‌it‌‌may‌‌be‌‌due‌‌to‌‌vasectomy‌‌operation.‌‌In‌‌such‌‌ 
cases,‌‌identification‌‌of‌‌semen‌‌from‌‌sperm‌‌is‌‌not‌‌possible.‌‌However‌‌immunological‌‌tests‌‌ 
using‌‌anti-semen‌‌sere‌‌against‌‌seminal‌‌plasma‌‌are‌‌increasingly‌‌being‌‌accepted‌‌as‌‌a‌‌labeled‌‌ 
test‌‌for‌‌aspermic‌‌semen.‌  ‌
 ‌
Chemically,‌‌semen‌‌is‌‌a‌‌complex‌‌mixture‌‌of‌‌organic‌‌and‌‌inorganic‌‌compounds.‌‌Important‌‌ 
constituents‌‌of‌‌semen,‌‌from‌‌an‌‌identification‌‌point‌‌of‌‌view,‌‌are‌‌proteins‌‌including‌‌ 
enzymes,‌‌blood‌‌group‌‌factors,‌‌choline,‌‌fructose,‌‌citric‌‌acid,‌‌uric‌‌acid,‌‌and‌‌zinc.‌‌The‌‌ 
composition‌‌varies‌‌from‌‌individual‌‌to‌‌individual.‌  ‌
 ‌
Visual‌‌location‌‌of‌‌semen‌‌is‌‌a‌‌difficult‌‌task‌‌because‌‌the‌‌stains‌‌are‌‌almost‌‌colorless‌‌and‌‌can‌‌ 
be‌‌destroyed‌‌by‌‌washing.‌‌Semen‌‌strains‌‌are‌‌found‌‌on‌‌the‌‌victim‌‌and‌‌the‌‌culprit‌‌on‌‌their‌‌ 
garments,‌‌at‌‌the‌‌scene‌‌of‌‌occurrence,‌‌and‌‌in‌‌the‌‌vehicle‌‌in‌‌one‌‌is‌‌used‌‌for‌‌the‌‌commission‌‌ 
of‌‌the‌‌crime.‌‌   ‌

Additional‌‌info:‌‌Saliva:-‌‌SALIVA‌  ‌
Saliva‌‌is‌‌a‌‌complex‌‌biological‌‌fluid‌‌secreted‌‌from‌‌acinar‌‌glands.‌‌Saliva‌‌samples‌‌can‌‌be‌‌ 
coupled‌‌with‌‌forensic‌‌in‌‌determining‌‌the‌‌DNA‌‌profiling‌‌of‌‌an‌‌individual‌‌and‌‌forensic‌‌ 
evidence.‌‌Samples‌‌of‌‌saliva‌‌and‌‌mouth‌‌swabs‌‌in‌‌the‌‌DNA‌‌testing‌‌is‌‌much‌‌easier‌‌than‌‌ 
blood,‌‌since‌‌the‌‌sample‌‌taking‌‌is‌‌easy,‌‌painless,‌‌and‌‌could‌‌be‌‌a‌‌form‌‌of‌‌test‌‌for‌‌children‌‌ 
also.‌‌No‌‌religious‌‌complications‌‌are‌‌also‌‌there‌‌which‌‌are‌‌present‌‌in‌‌the‌‌blood.‌‌It‌‌is‌‌found‌‌ 
to‌‌be‌‌an‌‌emerging‌‌forensic‌‌tool‌‌in‌‌cases‌‌of‌‌drug‌‌abuse,‌‌bite‌‌marks‌‌analysis,‌‌sex‌‌ 
determination,‌‌etc.‌  ‌

C.‌D
‌ NA‌‌PROFILING/‌‌FINGERPRINTING‌  ‌
 ‌
DNA‌‌fingerprinting‌‌(DNA‌‌typing,‌‌DNA‌‌identification,‌‌or‌‌genetic‌‌typing)‌‌is‌‌a‌‌technique‌‌ 
involving‌‌chemically‌‌dividing‌‌the‌‌DNA‌‌into‌‌fragments‌‌that‌‌form‌‌a‌‌unique‌‌pattern‌‌and‌‌ 
then‌‌matching‌‌that‌‌"identity‌‌profile"‌‌with‌‌the‌‌pattern‌‌obtained‌‌from‌‌similarly‌‌testing‌‌a ‌‌
suspect's‌‌blood‌‌specimen.‌‌If‌‌the‌‌two‌‌patterns‌‌match,‌‌the‌‌possibility‌‌of‌‌error,‌‌i.e.‌‌the‌‌ 
chance‌‌that‌‌they‌‌do‌‌not‌‌belong‌‌to‌‌the‌‌same‌‌individual‌‌may‌‌be‌‌less‌‌than‌‌one‌‌in‌‌30‌‌billion.‌‌ 
Dr.‌‌Alec‌‌Jeffreys‌‌in‌‌1985,‌‌developed‌‌DNA‌‌fingerprinting.‌‌   ‌
The‌‌human‌‌body‌‌consists‌‌of‌‌about‌‌six‌‌thousand‌‌billion‌‌cells‌‌which‌‌constitute‌‌tissue‌‌and‌‌ 
organ‌‌systems.‌‌Every‌‌living‌‌cell‌‌has‌‌genetic‌‌material‌‌contained‌‌in‌‌units‌‌called‌ 
chromosomes,‌‌which‌‌are‌‌located‌‌in‌‌the‌‌nucleus.‌‌DNA‌‌is‌‌present‌‌only‌‌in‌‌nucleated‌‌cells.‌‌ 
Each‌‌human‌‌somatic‌‌cell‌‌has‌‌23‌‌pairs‌‌of‌‌chromosomes.‌‌Gene‌‌is‌‌arranged‌‌along‌‌the‌‌length‌‌ 
of‌‌each‌‌chromosome.‌‌which‌‌are‌‌responsible‌‌for‌‌various‌‌functions.‌‌Each‌‌gene‌‌carries‌‌ 
instructions‌‌for‌‌the‌‌production‌‌of‌‌a‌‌particular‌‌protein‌‌that‌‌performs‌‌a‌‌particular‌‌function.‌‌ 
Genes‌‌are‌‌also‌‌responsible‌‌for‌‌the‌‌transmission‌‌of‌‌heredity.‌  ‌
 ‌
Following‌‌are‌‌the‌‌steps‌‌involved‌‌in‌‌DNA‌‌fingerprinting:‌  ‌

Isolating‌‌the‌‌DNA.‌  ‌
↓‌  ‌
Digesting‌‌the‌‌DNA‌‌with‌‌the‌‌help‌‌of‌‌restriction‌‌endonuclease‌‌enzymes.‌  ‌
↓‌  ‌
Separating‌‌the‌‌digested‌‌fragments‌‌as‌‌per‌‌the‌‌fragment‌‌size‌‌by‌‌the‌‌process‌‌of‌‌ 
electrophoresis.‌  ‌
↓‌  ‌
Blotting‌‌the‌‌separated‌‌fragments‌‌onto‌‌synthetic‌‌membranes‌‌like‌‌nylon.‌  ‌
↓‌  ‌
Hybridizing‌‌the‌‌fragments‌‌using‌‌labelled‌‌VNTR‌‌probes.‌  ‌
↓‌  ‌
Analyzing‌‌the‌‌hybrid‌‌fragments‌‌using‌‌autoradiography‌  ‌
There‌‌are‌‌two‌‌methods‌‌of‌‌DNA‌‌analysis‌‌in‌‌common‌‌use.‌‌(1)‌‌RFLP‌‌(restriction‌‌fragment‌‌ 
length‌‌polymorphism).‌‌(2)‌‌PCR‌‌(polymerase‌‌chain‌‌reaction).‌  ‌

1.‌‌Restriction‌‌fragment‌‌length‌‌polymorphism‌‌(RFLP)‌ 

1. The‌‌first‌‌step‌‌in‌‌this‌‌process‌‌is‌‌to‌i‌solate‌‌the‌‌DNA‌‌‌from‌‌the‌‌sample‌‌material‌‌to‌‌ 
be‌‌tested.‌‌The‌‌sample‌‌size‌‌for‌‌the‌‌RFLP‌‌test‌‌must‌‌be‌‌large‌‌enough‌‌to‌‌get‌‌the‌‌ 
proper‌‌result.‌  ‌
 2. Once‌‌the‌‌required‌‌size‌‌of‌‌the‌‌sample‌‌is‌‌available,‌‌the‌‌DNA‌‌is‌‌isolated‌‌from‌‌the‌‌ 
sample‌‌and‌‌is‌s‌ ubjected‌‌to‌‌restriction‌‌digestion‌‌‌using‌‌restriction‌‌enzymes.‌  ‌
3. The‌‌digested‌‌DNA‌‌sample‌‌is‌‌then‌s‌ eparated‌‌by‌‌agarose‌‌gel‌‌electrophoresis‌, ‌‌
in‌‌which‌‌the‌‌DNA‌‌is‌‌separated‌‌based‌‌on‌‌the‌‌size.‌  ‌
4. The‌‌next‌‌step‌‌is‌‌the‌t‌ ransfer‌‌of‌‌separated‌‌DNA‌‌from‌‌the‌‌gel‌‌slab‌‌onto‌‌the‌‌ 
nitrocellulose‌‌m ‌ embrane‌‌‌to‌‌hybridize‌‌with‌‌a‌‌labeled‌‌probe‌‌that‌‌is‌‌specific‌‌for‌‌ 
one‌‌VNTR‌‌region‌‌(radioactivity‌‌labeled‌‌complementary‌‌sequence‌‌for‌‌VNTR‌‌ 
region‌‌nucleotide‌‌sequence).‌  ‌
5. This‌‌technique‌‌of‌‌transferring‌‌and‌‌hybridizing‌‌DNA‌‌onto‌‌nitrocellulose‌‌ 
membrane‌‌is‌‌known‌‌as‌‌southern‌‌blotting,‌‌a‌‌most‌‌widely‌‌used‌‌DNA‌‌detection‌‌ 
technique‌‌by‌‌molecular‌‌biologists.‌  ‌
6. After‌‌the‌‌hybridization‌‌with‌‌the‌‌radioactive‌‌probes,‌‌the‌X ‌ -ray‌‌film‌‌‌is‌‌ 
developed‌‌from‌‌the‌‌southern‌‌blotting,‌‌and‌‌only‌‌the‌‌areas‌‌where‌‌the‌‌radioactive‌‌ 
probe‌‌binds‌‌will‌‌show‌‌up‌‌on‌‌the‌‌film.‌  ‌
7. Now‌‌these‌‌bands‌‌when‌c‌ ompared‌‌with‌‌the‌‌other‌‌known‌‌sample‌s,‌‌will‌‌give‌‌ 
the‌‌final‌‌result‌‌of‌‌the‌‌DNA‌‌fingerprinting.‌  ‌

Advantages‌  ‌
The‌‌RFLP‌‌is‌‌considered‌‌to‌‌be‌‌more‌‌accurate‌‌than‌‌the‌‌PCR,‌‌mainly‌‌because‌‌of‌‌the‌‌size‌‌of‌‌ 
the‌‌sample‌‌used‌‌more,‌‌the‌‌use‌‌of‌‌a‌‌fresh‌‌DNA‌‌sample,‌‌and‌‌no‌‌amplification‌‌ 
contamination.‌  ‌
Limitation‌  ‌
The‌‌RFLP,‌‌however,‌‌requires‌‌a‌‌longer‌‌time‌‌period‌‌to‌‌complete‌‌the‌‌analysis‌‌and‌‌is‌‌costly.‌  ‌
 ‌
2)‌‌POLYMERASE‌‌CHAIN‌‌REACTION‌‌(PCR):‌‌   ‌
This‌‌technique‌‌is‌‌used‌‌when‌‌a‌‌very‌‌small‌‌amount‌‌of‌‌DNA‌‌or‌‌a‌‌partially‌‌degraded‌‌ 
biological‌‌material‌‌is‌‌available.‌‌A‌‌small‌‌amount‌‌of‌‌DNA‌‌is‌‌amplified‌‌more‌‌than‌‌a ‌‌
million-fold‌‌using‌‌thermal‌‌TAQ‌‌polymerase.‌‌It‌‌is‌‌particularly‌‌useful‌‌for‌‌diagnostic‌‌ 
purposes.‌‌   ‌
 ‌
The‌‌PCR‌‌involves‌‌three‌‌major‌‌cyclic‌‌reactions:‌ 
 ‌
Denaturation‌  ‌
Denaturation‌‌occurs‌‌when‌‌the‌‌reaction‌‌mixture‌‌is‌‌heated‌‌to‌‌94℃‌‌for‌‌about‌‌0.5‌‌to‌‌2 ‌‌
minutes.‌‌This‌‌breaks‌‌the‌‌hydrogen‌‌bonds‌‌between‌‌the‌‌two‌‌strands‌‌of‌‌DNA‌‌and‌‌converts‌‌ 
them‌‌into‌‌single-stranded‌‌DNA.‌  ‌
The‌‌single‌‌strands‌‌now‌‌act‌‌as‌‌a‌‌template‌‌for‌‌the‌‌production‌‌of‌‌new‌‌strands‌‌of‌‌DNA.‌‌The‌‌ 
temperature‌‌should‌‌be‌‌provided‌‌for‌‌a‌‌longer‌‌time‌‌to‌‌ensure‌‌the‌‌separation‌‌of‌‌the‌‌two‌‌ 
strands.‌  ‌
 ‌
Annealing‌  ‌
The‌‌reaction‌‌temperature‌‌is‌‌lowered‌‌to‌‌54-60℃‌‌for‌‌around‌‌20-40‌‌seconds.‌‌Here,‌‌the‌‌ 
primers‌‌bind‌‌to‌‌their‌‌complementary‌‌sequences‌‌on‌‌the‌‌template‌‌DNA.‌  ‌
Primers‌‌are‌‌single-strand‌‌sequences‌‌of‌‌DNA‌‌or‌‌RNA‌‌around‌‌20‌‌to‌‌30‌‌bases‌‌in‌‌length.‌  ‌
They‌‌serve‌‌as‌‌the‌‌starting‌‌point‌‌for‌‌the‌‌synthesis‌‌of‌‌DNA.‌  ‌
The‌‌two‌‌separated‌‌strands‌‌run‌‌in‌‌the‌‌opposite‌‌direction‌‌and‌‌consequently‌‌there‌‌are‌‌two‌‌ 
primers-‌‌a‌‌forward‌‌primer‌‌and‌‌a‌‌reverse‌‌primer.‌  ‌
 ‌
Elongation‌  ‌
At‌‌this‌‌step,‌‌the‌‌temperature‌‌is‌‌raised‌‌to‌‌72-80℃.‌‌The‌‌bases‌‌are‌‌added‌‌to‌‌the‌‌3’‌‌end‌‌of‌‌the‌‌ 
primer‌‌by‌‌the‌‌polymerase‌‌enzyme.‌  ‌
This‌‌elongates‌‌the‌‌DNA‌‌in‌‌the‌‌5’‌‌to‌‌3’‌‌direction.‌‌The‌‌DNA‌‌polymerase‌‌adds‌‌about‌‌ 
1000bp/minute‌‌under‌‌optimum‌‌conditions.‌  ‌
The‌‌polymerase‌‌can‌‌tolerate‌‌very‌‌high‌‌temperatures.‌‌It‌‌attaches‌‌to‌‌the‌‌primer‌‌and‌‌adds‌‌ 
DNA‌‌bases‌‌to‌‌the‌‌single‌‌strand.‌‌As‌‌a‌‌result,‌‌a‌‌double-stranded‌‌DNA‌‌molecule‌‌is‌‌ 
obtained.‌  ‌
 ‌
These‌‌three‌‌steps‌‌are‌‌repeated‌‌20-40‌‌times‌‌in‌‌order‌‌to‌‌obtain‌‌a‌‌number‌‌of‌‌sequences‌‌of‌‌ 
DNA‌‌of‌‌interest‌‌in‌‌a‌‌very‌‌short‌‌time‌‌period.‌  ‌
Advantages:‌A ‌ ‌‌small‌‌amount‌‌of‌‌specimen‌‌is‌‌sufficient‌‌for‌‌the‌‌test.‌‌Takes‌‌a‌‌shorter‌‌time‌‌ 
to‌‌complete.‌‌Less‌‌costly.‌  ‌
 ‌
Limitation:‌L ‌ ess‌‌accurate‌‌than‌‌RFLP,‌‌Possibility‌‌of‌‌amplification‌‌contamination.‌  ‌
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