UNIT 1 BLOOD GROUP TYPING: ABO AND

RH (D) BLOOD GROUPS

Contents
1.0 Introduction
1.1 ABO Blood Group System
1.2 Rh Blood Group System
1.3 Phenotyping of ABO and Rh Blood Groups
1.3.1 Materials Required
1.3.2 Preparation of 3% RBC
1.3.3 Phenotyping of ABO and Rh Blood Groups
1.3.4 Detection of Weak D Phenotype
1.3.4.1 Materials Required
1.3.4.2 Procedure 1.4

References
Learning Objectives
After reading this unit, you will be able to:
 Elucidate about the importance of ABO and Rh blood groups in blood
transfusions; and
 Acquire knowledge of phenotyping of ABO and Rh blood group system
involving forward and reverse phenotyping

1.0 INTRODUCTION

Determination of blood group is called blood group typing. Blood groups are
red blood cell (RBC) antigen containing blood systems. Blood groups are
determined using RBCs. As per the International society of Blood Transfusion
(www.isbtweb.org) report dated 30th June, 2021, 43 blood groups have been
LGHQWL¿HGLQKXPDQV,QIRUPDWLRQRIEORRGJURXSVLVXVHGI
RUYDULRXVDSSOLFDWLRQV such as, as a tool for genetic relationship
between populations and migration, and to investigate the population
characteristics and diversity in populations.
,QYHVWLJDWLRQVRIKXPDQYDULDWLRQDLGLQWUDFLQJRULJL
QPHGLFDOO\VLJQL¿FDQW variants and to eliminate racial prejudices.
Blood group typing is based on the principle of agglutination (clumping) of
RBC containing antigen (for example: A or B or AB or Rh (D)) when react with
antibody (for example: anti-A or anti-B or anti-AB, anti Rh (D)) present in the
plasma, broadly indicating interaction of antigen and antibody. Clumping of
RBC indicates the presence of respective blood group. Each individual carries
only one type of antigen and antibody for a particular blood group.

173
Practical Manual

Antigen is any substance that induces an immune response. In case of blood

groups, antigens are polysaccharide or proteins in nature which are encoded by

Contributed by Dr. SAA Latheef, Department of Genetics and Biotechnology, Osmania University,
Hyderabad
genes located mostly on autosomes or sex chromosomes (X or Y). Antibody is
a protein produced by plasma cell, a differentiated B lymphocyte in response to
immune response induced by antigen. Blood group antibodies (natural
occurring substances) found on immunoglobulin M molecules in plasma are
formed in the
¿UVW\HDURIKXPDQOLIHZKHQH[SRVHGWRIRRGEDFWHULDDQG
YLUXV,QODERUDWRULHV for teaching purpose, blood groups are
determined using voluntary donor RBCs and commercial antibodies. In blood
banks, before blood transfusion, blood group typing is done for both the donors
and recipients/patients and also using RBC of donor with serum of recipient, a
procedure known as cross matching. Depending on the need of type of blood
component (RBC, plasma and platelets) transfusions of particular blood
component are carried out. With respect to blood transfusions in humans, 9
blood groups (ABO, Rh , MNS, Kidd, Kell, P, Lewis, Duffy and Lutheran) are
important for the purpose of blood component transfusions. For the present unit,
determination of ABO and Rh blood groups are described.

1.1 ABO BLOOD GROUP SYSTEM

In 1901, Karl Landsteiner discovered A, B, O of ABO blood group system while

the AB blood group was discovered by de Castello and Steini in 1902. For the
discovery of A, B and O blood groups of ABO system, Karl Landsteiner was
awarded Nobel Prize in Physiology or Medicine in the year 1930. The gene for
ABO system is located on the chromosome 9.

This system has four blood groups (A, B, AB and O) which are determined
based on the presence and absence of antigens and antibodies. Antigens and
antibodies are present in the blood (the former present on the red cells and the
latter in blood plasma). The antigens are designated as A and B and the
antibodies as anti-A and anti-B.

Group A person carries antigen A and antibody anti-B. Group B person carries
antigen B and antibody anti-A. Group O individuals possess both the antibodies
and lack any antigen while group AB individuals carry both the antigens but
lack any antibody.

Individuals with O blood group can donate any blood component (RBC, plasma,
and platelets) to individuals carrying all blood groups. Carriers of A blood group
can donate any blood component to either A or AB; carriers of B blood group
can donate blood components to the carriers of B and AB and individuals of AB
blood group can donate blood components to the carriers of AB blood group. In
174
other words, we can understand this as carriers of A blood group can receive
any blood component from carriers of A as well as O blood groups. Carriers of
B blood group can receive the blood components from B as well as O blood
groups. Carriers of O blood group can receive blood components only from O
blood group, while carriers of AB blood group can receive blood components
from all blood groups, A, B, AB and O. On the basis of the above explanations,
we can conclude that while blood group O (-ve) is a universal donor, blood
group AB (+ve) is a universal recipient.
Three genes namely A, B, and O controls the system. A and B allele are
codominant and both A and B are dominant to O which is recessive. Inheritance
of ABO follows Mendelian pattern. In World population the frequency of blood
group O (63%) is dominant followed by A (21%) and B (16%). Indian
populations are characterized by predominance of ‘B’ blood group
(Venkatramana, 2012). Among Europeans, the Blood group ‘A’ is dominant, ‘B’
in Asia and ‘O’ in South America, Siberia and some areas of Switzerland
(Farhud and Yeganeh,2013). In
,QGLD¿UVWLQYHVWLJDWLRQVRQEORRGJURXSVZHUHGRQHRQ
VROGLHUVE\+LUVFK¿HOG in 1919. ABO blood group is a good example
of multiple alleles which was
GLVFRYHUHGE\)HOL[%HUQVWHLQLQ$%2EORRGJURXSZDVWKH¿
UVWVWXGLHG polymorphism in humans and it was the data of this marker
that was used for the construction of evolutionary tree.

Blood groups were also studied for susceptibility to diseases among its carriers.
For example, it was observed that carriers of ‘O’ blood group were found to
have higher risk of developing gastric and duodenal ulcer, cholera and other
types of diarrhoea than carriers of other blood groups. Among carriers of A and
B blood groups, more B than A blood groups carriers were found to be
susceptible to cholera. Carriers of A and AB were shown to be at risk of getting
infected with small pox than people carrying other blood groups. Carriers of A
blood group were shown to be susceptible to gastric cancer (Sarkar, 2012; Dean,
2015). In forensic labs using blood groups, paternity disputes are resolved and
suspected
FULPLQDOLGHQWLW\LVFRQ¿UPHG

1.2 RH BLOOD GROUP SYSTEM

Rh blood group was discovered by Karl Landsteiner and Weiner in 1940.This

blood group got its name because of discovery of Rh factor. Discoverers of this
blood group injected RBC of Rhesus monkey into the Rabbit and after a series
of injections, plasma of immunized Rabbits caused clumping of RBC of not
only Rhesus monkey and but also of humans. Since then, Rh factor name was
retained for Rh (D) antigen. This blood group also came into limelight due to
its association with Erythroblastosis fetalis, a haemolytic disease of the new
born. This occurs due to the production of IgG antibodies, in plasma of Rh-

175
Practical Manual

(negative) mother against D antigen of foetus, which enter through placenta

during labour into the foetus and causes haemolysis, leads to anaemia and death
of the foetus, mostly in second pregnancy. Rh blood group is a complex blood
group in humans and contains 56 antigens (www.isbtweb.org). Among them the
notable are D, C, E, c, and e (Dean, 2015). These antigens are encoded by RHD
and RCHE genes harboured on chromosome 1. RHD gene encodes RHD
antigen whereas RHCE gene encodes CE antigens in varying combinations such
as Cece, CE or cECx (RH9), Cw (RH8) and VS (RH20). Carriers of Rh (D)
antigen are called Rh+ (positive) and those lacking it are labelled as Rh-
(negative). Rh+ is predominant in Asians and Rh- in Caucasians. C and E
antigens are predominant in Asians; c and e antigens in Blacks and Caucasians
(Dean, 2015). Rh antigens are only expressed on RBC only.

1.3 PHENOTYPING OF ABO AND RH BLOOD

GROUPS

Phenotyping or determination of blood groups is carried out on porcelain tile/

glass slide/ microcentrifuge tube /microplate. Phenotyping of blood groups is
done using commercially available antisera.

1.3.1 Materials Required

1) 3% Red Blood Cells (weight/volume) (W/V)
2) 0.9% (W/V) saline
3) Labtop centrifuge
4) Polystyrene/glass tubes (5mL)
5) Micro/mini centrifuge
6) Microcentrifuge tubes (1mL)
7) Microcentrifuge tubes (2mL)
8) Micropipettes (100µl and 1mL)
9) Micropipette tips (100µl and 1mL)
10) Commercial antibodies (antisera-A, B, H and D)
11) Microcentrifuge tube stand
12) Double distilled water
13) Glass beaker
14) Glass bottle

176
 1.3.2 Preparation of 3% RBC
1) A 5mL venous blood is drawn from human volunteer and the drawn blood
is slowly released through the walls of 5mL falcon tube or glass tube
containing anticoagulant (sodium citrate (160mg) or Ethylene diamine tetra
acetic acid (10mg) or heparin (75IU/mL) and mixed well by rolling the tube
gently on palm or bench for 5 to 6 minutes.
2) Centrifuge the falcon/glass tube at 1000 revolutions per minute (rpm) in
labtop centrifuge.
3) The supernatant formed in falcon/glass tube is collected into the 2mL
microcentrifuge tubes.
4) 100 mL of 0.9% saline (W/V) is prepared by adding 900mg of sodium
Chloride (NaCl) into a glass beaker containing 100mL of double distilled
water and store in glass bottle at room temperature.
5) To the falcon/glass tube from which supernatant is removed, add 3.5mL of
0.9% saline with 1mL micropipette.
6) Centrifuge the falcon/glass tube at 2500 rpm for 3 minutes in labtop
centrifuge. Repeat this step several times till the supernatant in falcon/glass
tube appear as transparent.
7) For preparing 2mL volume of 3% RBC, 60µl of RBC suspension is added
to the 2mL microcentrifuge tube containing 1940µl of 0.9% saline using
either 1mL or 100µl micropipette.
8) Mix the contents in 2mL microcentrifuge tube with 1 mL micropipette
several times by aspirating and dispensing into the microcentrifuge tube.

Fig. 1.1: Labtop Centrifuge

(Source: https://www.biomall.in/product/labtop-centrifuge-lrc 25r-lrc-25r)

177
Practical Manual

Fig. 1.2: Micro or mini centrifuge

(Source: https://www.pipette.com/Microcentrifuges)

Fig. 1.3: Polystyrene 5 mL tubes

(6RXUFHKWWSVZZZLQWVFLHQWL¿FFRPSURGXFWSEW)

Fig. 1.4: Glass tubes (5mL)

(Source: https://www.universalmedicalinc.com/13mm-x-75mm-5ml-test-tubes.html)

178
Fig. 1.5: Variable volume micropipette
(Source: https://ien.labbox.com/product/variable-volume-micropipette-easy-40-elite/)

Fig. 1.6: Micropipette tips in boxes

(Source: https://indolabutama.com/catalogue/pipette-tips/)

Fig. 1.7: Microcentrifuge tubes (Source: Starlab.com)

1.3.3 Phenotyping of ABO and Rh Blood Groups

1) Take 4 microcentrifuge tubes (1mL) and label them as A, B, H and D.
2) Add 100µl of antisera- A, B, H and D into each of 4 microcentrifuge tubes.
3) Add 100 µl of 3% RBC suspension to each of 4 microcentrifuge tubes
4) Centrifuge the microcentrifuge tubes for 1 minute at 1000 rpm in
minicentrifuge
5) Formation of agglutination of RBC in microcentrifuge tubes indicates the
presence of particular blood group. Results are interpreted as shown in
Table 1.1.
If there is an agglutination in microcentrifuge labelled A, it is A blood group; if
there is an agglutination in the microcentrifuge tube labelled B, it is B blood
group; if agglutination found in both A and B labelled microcentrifuge tubes
then it is considered as AB blood group and if there is agglutination in the
tube labelled H, it is O group.
179
Practical Manual

If agglutination found in microcentrifuge labelled D, indicated presence of

Rh+ (positive) blood group.

Big clump (agglutination) 2 clumps

Small clumps small clump

Fig. 1.8: Intensity of Agglutination (Source: NIB, Guidance manual, 2013)

Table 1.1: Interpretation of ABO System Results
ABO Blood group

Anti-A Anti-B Anti-H Result

Agglutination present Agglutination Agglutination A blood group
absent absent
Agglutination absent Agglutination Agglutination B blood group
present absent
Agglutination present Agglutination Agglutination AB blood group
present absent
Agglutination absent Agglutination Agglutination O blood group
absent present
Rh blood group

Anti-D

Agglutination present Rh positive

Agglutination absent Rh negative

1.3.4 Detection of Weak D Phenotype

,IDSDUWLFXODULQGLYLGXDOLVLGHQWL¿HGDV5KQHJDWLYHW
KHUHLVDSRVVLELOLW\RI
SUHVHQFHRIZHDN''XSRVLWLYHDQWLJHQLQWKDWLQGLYLGXD
O7RFRQ¿UPLI weak D (Du)+ antigen is present, indirect agglutination test is
performed.

1.3.4.1 Materials Required

1) Anti human globulin
180
2) 0.9% saline
3) Minicentrifuge/Microcentrifuge
4) Micropipette (1mL)
5) Micropipette tips (1mL)
6) Water bath
7) Microcentrifuge tube stand
8) Microscope
1.3.4.2 Procedure
1) Incubate the microcentrifuge tube (1mL) determined as Rh-(negative) as
per procedure described in 1.3.3 at 37ºC in water bath for 30minutes.
2) Centrifuge the microcentrifuge tube for 1 minute at 1000 rpm in
minicentrifuge.
3) If agglutination of RBC is seen, consider it as Rh+ (positive). If no
agglutination, proceeds to further steps.
4) Add 800µl of 0.9% saline into Rh- containing microcentrifuge tubes using
1mL micropipette and centrifuge the microcentrifuge tube at 1000rpm in
minicentrifuge and remove the supernatant (liquid). Repeat this step three
times.
5) Add 200 µl of anti human globulin to the microcentrifuge tube with 1mL
pipette.

*HQWO\VKDNHWKHPLFURFHQWULIXJHWXEHZLWKWDSSLQJ
WKH¿QJHUDQGH[DPLQH for the presence of agglutination of RBC
under the light or microscope.
7) If agglutination of RBC is seen in microcentrifuge tube, the presence of
ZHDN''XLVFRQ¿UPHGDQGLQWKHDEVHQFHRILWLWLVGHWHU
PLQHGDV5K (negative).

181
Practical Manual

Fig. 1.9: Water bath

(Source: https://en.wikipedia.org/wiki/Laboratory_water_bath#/media/File:Shaking_water_bath_2015.
JPG)

Fig. 1.10: Microscope

(Source: https://www.quirumed.com/ie/professional-binocular-microscope-achromatic.html)

182