Unit 11

Blood and other body fluid evidences

Blood as physical evidence
Blood is a vital component of the circulatory system, consisting of plasma and cells (red blood cells,
white blood cells, and platelets). It transports oxygen, nutrients, and waste products throughout the body.
When blood is removed from the circulatory system, it clots, forming a solid coagulum and a clear liquid
called serum. Blood is important in forensic investigations, as its presence, clotting patterns, and DNA
can provide crucial evidence about the time of injury, position of the victim, and nature of the incident.
Blood analysis helps in identifying individuals and understanding crime scenes.

Composition of blood

Blood consists of plasma (55%) and formed elements (45%). Plasma is a liquid solution
containing 10% dissolved substances, including plasma proteins (7%), inorganic salts (0.9%),
and organic compounds like amino acids, vitamins, hormones, and lipoproteins. The formed
elements include:

 Red Blood Cells (RBCs): These nucleus-free cells are filled with hemoglobin, which
binds oxygen and gives blood its red color. Hemoglobin forms oxyhemoglobin in
oxygen-rich areas and releases oxygen in low-oxygen regions. RBCs also contain
carbonic anhydrase, aiding CO₂ transport. RBCs are typically round and biconcave,
except in camels, where they are oval.
 Platelets: Involved in blood clotting.
 White Blood Cells (WBCs): Part of the immune system.

RBC size varies among species, ranging from 5–10 µm in mammals. Interestingly, the size of
RBCs does not correlate with the animal's size, as seen in mice and elephants having similar
RBC sizes.

Interaction of Antibody with Antigen

An antibody is an immunoglobulin produced by lymphoid cells in response to exposure to an

antigen, which can be harmful (like bacteria or viruses) or harmless (like foreign proteins).
Antibodies bind specifically to antigens that are identical or nearly identical to the one that
triggered their production. The interaction occurs when the antibody's binding site forms a link
with the antigenic determinant of the antigen. The strength of this bond, or affinity, depends on
how well the antibody's binding site fits the antigenic determinant. Antibodies vary in their
combining quality, and their overall ability to bind with antigens can be quantified
experimentally. Studies show that antibody affinity increases with repeated immunization and
that antigen dose can influence antibody quality.
SPECIES IDENTIFICATION

Species Identification involves the interaction of antibodies and antigens to form complexes that
may precipitate. Precipitation is optimal when antigen and antibody are in ideal proportions,
forming large aggregates visible as precipitates. In antibody excess, little linking occurs, and in
antigen excess, fewer complexes form. This reaction is useful in forensic studies and food
adulteration detection. A common modification uses agar gel, where antigen and antibody
diffuse, creating a concentration gradient. Precipitin bands form where optimal proportions of
antibody and antigen meet, allowing identification of unknown antigens based on band patterns.

Blood grouping

Blood grouping has advanced significantly over the past century. Initially, it was impossible to
distinguish between human and animal blood, and blood could not be classified into groups (A,
B, AB, O). Early tests by Uhlenhuth and Landsteiner improved identification, but it wasn't until
DNA studies that individual identification became highly reliable. Agglutination is a key
technique in blood grouping, where antibodies bind to antigens on red blood cells, causing
visible clumping (agglutination). For example, anti-A serum agglutinates A-type cells but not B
or O. The test is also used in hormone detection, where agglutination is blocked by free hormone
molecules. Agglutination tests can be quantitative by comparing unknown samples with known
standards.

The Discovery of ABO Blood Groups

The ABO blood group system was discovered by Karl Landsteiner in 1901. Before this,
transfusions were risky due to the incompatibility of blood types. Landsteiner observed that
mixing the serum and red blood cells of different people caused agglutination (clumping),
leading him to identify three distinct blood groups: A, B, and C (later renamed O). In 1902,
groups A, B, AB, and O were formally defined.

 Group A: Has A antigens on red cells and anti-B antibodies.

 Group B: Has B antigens and anti-A antibodies.
 Group AB: Has both A and B antigens and no anti-A or anti-B antibodies.
 Group O: Has no A or B antigens, but has both anti-A and anti-B antibodies.

Landsteiner’s discovery revolutionized blood transfusions and immunology by highlighting the

need for blood compatibility.

 The Rhesus (Rh) Blood Group System is the second most important after the ABO
system. It involves antigens such as C, c, D, d, E, and e, with D being the most significant
for transfusions. Around 85% of Caucasians and 94% of Negroes are Rh-positive. The
Du variant of the D antigen exists, where cells carrying it are not agglutinated by routine
anti-D sera but are considered Rh-positive for donors and Rh-negative for recipients.
 The MN Blood Group System was described by Landsteiner in 1927 and categorizes
individuals into MM, MN, and NN genotypes based on antigens M and N. The use of
anti-sera for forensic testing is recommended with pooled erythrocytes.
  The Lewis Blood Group System involves naturally occurring antibodies against Lewis
antigens, which are soluble and not part of the red blood cells. These antigens can
sometimes cause hemolytic transfusion reactions when absorbed onto red cell surfaces.
 Paternity Testing and Blood Grouping: Blood grouping can provide evidence in
paternity cases, as it shows possible and impossible father-child relationships based on
blood types. For example, if the mother is blood group O and the child is AB, the father
cannot be O.
 Sample Collection and Handling: Proper handling of blood samples is crucial for
forensic analysis. Wet bloodstains should be stored in a test tube and kept on ice, while
dry stains should be carefully packaged in envelopes. Small objects with blood (e.g.,
knives) should be transported with care to prevent contamination. Larger substrates (e.g.,
floors) may require cutting a portion for analysis. It's important to keep samples dry and
refrigerated to prevent bacterial growth and preserve blood group elements.

Methods for Bloodstain Identification:

 Benzidine Test: This test detects blood by catalyzing oxidation of benzidine in the
presence of hydrogen peroxide, producing a colored compound. The sensitivity is around
1:200,000. However, it is nonspecific, as it can also react with other oxidizing
compounds like potassium permanganate, garlic, and vegetables.
 Phenolphthalein Test: More sensitive than the Benzidine test (1:1,000,000), it is also
nonspecific and reacts with substances like copper and potassium ferricyanide. It's used
as a preliminary test, and simultaneous use of both tests can reduce errors. Control tests
should be conducted without hydrogen peroxide to avoid interference.
 Crystal Test: Hemoglobin derivatives such as hemin, hematin, and hemochromogen are
identified through crystal formation and microscopic examination, providing a highly
reliable method for confirming bloodstains.
 Ouchterlony Double Immunodiffusion: This method is used for species identification
by comparing the reaction of an unknown protein with a known protein against a specific
antiserum.
 The Holzer method is used in forensic biology to determine blood groups from dried
bloodstains using known positive and negative controls. Blood analysis in forensic
investigations helps reconstruct crime scenes, link suspects to victims, and identify
weapons. DNA technology now replaces traditional serology methods like ABO
grouping for more accurate individual identification. Bloodstain patterns, fingerprints,
and tissue evidence provide crucial clues to solve crimes and verify alibis, making
forensic biology an essential tool in criminal justice.

Seminal fluid

Examination of seminal fluid is crucial in medico-legal investigations, especially in cases of

rape, sexual homicide, or adultery. The presence of spermatozoa is the most definitive proof.
Fresh specimens yield the best results, as sperm cells may be found in the vaginal area, vulva, or
pubic hair if the victim is examined promptly. In cases of delayed investigation, careful
examination of the genital region is necessary. Immediate collection and microscopic analysis of
fresh specimens are essential for successful identification of seminal fluid.