Blood Grouping and Rh Typing

Aim:
To determine one’s own blood group through blood grouping and Rh typing.
Principle:
The principle of blood grouping and Rh typing is based on the discovery that
human blood is divided into four groups: A, B, AB, and O. These groups are
determined by the presence or absence of antigens A and B on red blood cells
(RBCs). Antigen A or B triggers the corresponding antibody (anti-A or anti-B) to
stick to RBCs with the opposing antigen. RBCs with antigens A and B never occur
together, preventing them from sticking together. This principle also states that
blood plasma contains antibodies that correspond to any antigens not present on
RBCs. For example, blood group A has antigen A on RBCs and anti-B in plasma,
while blood group O has no antigens on RBCs but has both anti-A and anti-B
antibodies in plasma.
Materials Required:
- Glass slide with three cavities
- Glass marker
- Sterilized needle
- 75% ethanol
- Cotton
- Antiserum A
- Antiserum B
- Antiserum D (Rh factor)
- Toothpicks
- Small glass tube
- Micropipette
Procedure :
1. Begin by cleaning the tip of the left middle finger with 75% alcohol and
allowing it to dry.
2. Sterilize a needle and prick the tip of the left middle finger to draw blood.
3. Collect a drop of blood using the small glass capillary tube.
4. Place a drop of blood in the glass slide after marking beneath the glass slides
with 3 circles. Mention as A, B, D.
5. Label one circle as “A” and the other as “B” using the glass marker.
6. Add a drop of Anti-A serum to the “A” marked circle and Anti-B serum to the
“B” marked circle.
7. Mix the blood and serum in each cavity using separate sterilized toothpicks.
8. To determine Rh typing, repeat steps 5-7 using Anti-D serum in a separate cavity
labeled “Rh”.
9.This procedure allows for both blood grouping and Rh typing using the same set
of materials and steps.
Result: (Blood Grouping)
(Note: Select the result according to your blood group)
- If agglutination occurs with Anti-A serum, the blood type is A.
- If agglutination occurs with Anti-B serum, the blood type is B.
- If agglutination occurs with both Anti-A and Anti-B sera, the blood type is AB.
- If there is no agglutination with either serum, the blood type is O.
Result: (Rh typing)
- If agglutination occurs, the blood type is Rh positive.
- If there is no agglutination, the blood type is Rh negative.
 Separation of Serum and Plasma from Blood
Aim:
To separate serum and plasma from blood samples for various laboratory
applications.
Principle:
When blood is allowed to clot completely, the clear liquid that separates is called
serum. Plasma, on the other hand, is obtained from blood treated with an
anticoagulant such as EDTA. Both serum and plasma are vital components used in
various laboratory experiments, especially in immunological studies and testing.
Materials Required:
- Needles
- EDTA (Ethylenediaminetetraacetic acid)
- Freshly collected human blood
- Cotton
- Centrifuge tubes
- Micropipette
Procedure:
1. Blood Collection:
- Use a sterile needle to collect blood into a test tube.
- For serum separation, allow the blood to clot at room temperature for
approximately 1 hour.
- For plasma separation, add EDTA to the blood sample immediately after
collection and mix gently. EDTA prevents clotting.
2. Serum Separation:
- After clotting, gently remove the clotting material using a clean glass rod to
avoid hemolysis of red blood corpuscles.
 - Refrigerate the sample to facilitate further separation.
- Decant the serum delicately to avoid disturbing the clot, and transfer it to a
centrifuge tube.
- Centrifuge the tube at 3000 rpm for a specified time at room temperature.
- Store the obtained serum, which can also be lyophilized for preservation.
- Use commercially available preservatives to prevent bacterial and fungal
growth, and store samples at 4°C in small volume tubes (0.1 to 0.5ml).
3. Plasma Separation:
- Place the blood sample treated with EDTA in a centrifuge tube.
- Centrifuge the tube at under 3000 rpm to separate the plasma from other blood
components.
- Collect the supernatant, which is the plasma, and store it at -20°C.
- Plasma can be used for various applications after separation.
Result:
Serum and plasma have been successfully separated from the blood samples and
stored in the refrigerator for further use.

SPOTTERS:
1)RIA
1.RIA- Radioimmunoassay
2. RIA is highly sensitive and specific technique that can detect very low levels of
a target molecule in a sample.
3.This makes it useful for measuring the concentration of hormones, cytokines, and
other small molecules in biological fluids like blood and urine.
4.RIA has been extensively used in immunology research to measure the levels of
cytokines and other signalling molecules involved in the immune response.
5.It has also been used to measure the levels of antibodies in patients samples to
diagnose and monitor autoimmune diseases.
6.This can make the technique more expensive and time consuming compared to
other assays, such as ELISA

2)ELISA (Enzyme-Linked Immunosorbent Assay)

1.Enzyme-linked immunosorbent assay (ELISA) is a widely used immunological
assay that is used to detect and quantify the presence of specific antigens or
antibodies in a sample.
2.ELISA works by using a solid phase (such as microtiter plate) that has been
coated with either the antigen or antibody of interest.
3.The sample is added to the solid phase and any specific antigen or antibody will
bind to the coated surface.
4.ELISA is a highly sensitive and specific assay that has numerous applications in
clinical and research settings.
5.It can be used to diagnose infections, detect antibodies in vaccine trials and
monitor the progression of diseases such as cancer and autoimmune disorders.