USER MANUAL

Blood Grouping Kit

Determination of blood group

Catalog No: IMI-KIT-1014 – for 100 experiments

Duration: 10 minutes

Blood grouping Kit contains

Standard reagents
and
Protocol

An ISO 9001:2000 Certified Company

IMGENEX INDIA PVT. LTD.

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Blood Grouping Kit User manual

User manual of blood grouping

I. PRINCIPLE

The ABO blood grouping system was first developed around 1900 and till now the major human blood
group system. All individual can be placed into one of four major groups: A, B, AB or O. ABO blood
grouping is based on the presence or absence of two blood group antigens found on the red blood cell
surface. These antigens are designated as A and B and are derived from inherited genes. A person
having A antigen belongs to Group A; having B antigen belongs to Group B; having both A and B
antigens belongs to Group AB; and a person having neither A nor B antigen belongs to Group O.

Human blood also contains blood group antibody that reacts with corresponding blood group antigen. An
antibody that reacts with antigen A is called anti-A antibody and an antibody that reacts with antigen B is
called anti-B antibody. Blood group antibodies naturally occur in human serum. Group A individuals do
not have anti-A antibody and Group B individuals do not have anti-B antibody. Accordingly Group AB
individuals do not have either anti-A or anti-B antibody and Group O individuals have both anti-A and anti-
B antibodies.

The RhD antigen is another important aspect in determining blood type. The term "positive" or "negative"
refers to either the presence or absence of the RhD antigen. Anti-RhD antibody is not usually a naturally
occurring antibody as the Anti-A and Anti-B antibodies are.

The ABO blood grouping procedure is based on the principle of agglutination or clumping. The blood of
the patient is reacted with known antibodies (anti-A, anti-B and anti-D separately) and the agglutination
reaction is observed. If the antigen present in the blood cell corresponds to the antibody, it causes
agglutination or clumping. If the blood is agglutinates with anti-A antibody, the blood group is A and if it
agglutinates with anti-B, the group is B. On the other hand, if agglutination is seen with both anti-A and
anti-B, it is Group AB and if there is no agglutination it is Group O.

While blood grouping if the blood agglutinates in presence of anti-RhD antibody, it is denoted as positive
and if the blood does not agglutinate, it is denoted as negative.

II. OVERVIEW
Determination of blood group is extremely important in blood transfusion medicine and yet very easy to
perform. The patient’s blood is reacted with anti-A, anti-B and Anti-RhD antibodies separately and the
agglutination reaction can be visible in naked eye to determine the blood group.

III. ADVANTAGES
• This kit helps the student to understand the principle and basic function of the technique.
• The technique is relatively simple, rapid to perform and of low cost because it requires no special
equipment.
• It contains standard reagents and protocol for better results.

IV. KIT COMPONENTS AND STORAGE

All the reagents should be stored at 2-8°C when not in use.

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Blood Grouping Kit User manual

Sl No Components Quantity (100 tests)

1 anti-A sera 5 ml

2 anti-B sera 5 ml

3 anti-RhD sera 5 ml

4 Microscopic slide 10

5 Wax pencil 1

6 Needle (24G) 100

7 Instruction manual 1

Materials Required (not included in the kit):

Cotton, 70% alcohol, toothpicks.

V. PROCEDURE:

1. Take one microscopic slide, clean and dry it. The slide must be very clean so it does not interfere
with the reaction.
2. With a wax pencil, draw two lines on one surface of the slide to divide the surface into three parts
and draw three circles, one in each part as shown in Figure1.

A B Rh

Figure 1: Template on a glass slide for blood grouping.

3. Place one drop of the corresponding antiserum (at room temperature) near the edge but within
each of the circles as shown in Figure 2.

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Blood Grouping Kit User manual

4. Choose a little finger (usually left ring finger). Clean this fingertip with an alcohol in a cotton ball
and let it air dry. Keep the cotton ball nearby, as it needed again. Dangle the hand down to
increase the amount of blood in the fingers.

A B Rh

Figure 2: Addition of anti-sera on the glass slide.

5. Press on the bottom of the fingertip with the thumb of the same hand (to help hold blood in the
fingertip) and quickly prick the fingertip with the help of a needle.
Note: The needle is sterile, so do not touch the tip with anything before using it.
6. Quickly, let one drop of blood into each circle but not touching the anti-sera yet. Do not touch any
of the anti-sera.
7. After putting three drops of blood, apply gentle pressure to the wound with cotton ball.
Remember to properly dispose the used needle.

A B Rh

Figure 3: Blood added on the glass slide.

8. Use a tooth toothpick to mix the blood and antiserum and stir gently. Do it for each of the circles
using a fresh toothpick every time. The wax pencil circle will help to keep the sample isolated.
9. Watch to see if any of the samples show agglutination. The agglutination will appear as the grainy
clumps of red blood cells (RBCs) suspended in a clear solution. Rh is slower to agglutinate, so do
not give up too soon.

Result and inference: Determine the blood type depending on the result. Following table can be used to
determine the blood type:

Table: Determination of blood group (type).

Anti-A Anti-B Type

Yes No A
No Yes B
Yes Yes AB
No No O

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Blood Grouping Kit User manual

Independent of whether agglutination occurs in anti-A and anti-B sera, clumping may or may not occur in
anti-RhD serum. If agglutination occurs in anti-RhD serum, the Rh factor is positive; and if it does not, the
Rh factor is negative.

VII. REFERENCES
1. Yamamoto F et al. Nature. 345:229-233 (1990).
2. Daniels G. Human Blood Groups, Second ed., Blackwell Science (2002).
3. Reid ME and Lomas-Francis C. The Blood Group Antigen Facts Book. Second ed., Elsevier
(2004).

VIII. TROUBLESHOOTING

Problem Probable Cause Suggestion

Need not to worry. You have O-
No agglutination No antigen
type blood group.

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