BLOOD GROUPING &

TRANSFUSION
- Dr Kaberi De
Blood Groups and Physiological Basis of Blood Transfusion

• BLOOD GROUPS- Human red cells contain numerous surface structures that are recognized as
antigens by the immune system of the individual. These surface structures are called blood
group antigens. Based on the presence of antigens on red cell membrane, usually
corresponding antibodies are absent in the plasma of that individual. This forms the
physiological basis of blood transfusion
• The blood group antigens are called agglutinogens and the antibodies against them are called
agglutinins as the reaction between them results in clumping or agglutination of red cells.
Blood groups and principle of blood transfusion:
• There are more than 30 known blood group systems in our blood containing about 400
antigens.
• The blood group systems that are most important for blood transfusion are ABO and Rh
systems.
• Other major blood group systems that have medical and medicolegal importance are MNS,
Lewis, Duffy, Kell, P and Lutheran systems.
 Uses of Blood Groups
• Red cells have traditionally been considered as less active cells that contain mainly hemoglobins. However,
they play important role in many physiological processes of the body due to the presence of antigens on
their surface:
• Characterization of red cell antigen and antibodies forms the basis of compatibility testing for blood
transfusion. When the blood group of an individual is known, in emergency situations, blood can be
transfused immediately with the suitable blood. Therefore, ABO and Rh blood groups are usually displayed
in the identity cards of individuals.
• The blood group antigens have provided the scientific basis for understanding hemolytic diseases of the
newborn and autoimmune hemolytic anemias.
• Blood group antigens play a critical role in susceptibility to infections by malaria parasites, bacteria and virus
• Red cell antigens are also associated with clinical disorders. Absence of certain red cell antigens is associated
with specific dysfunctions, and certain inherited and acquired diseases are associated with alteration of red
cell antigen expression. These associations have led to understanding of pathophysiology of these
pathologic processes.
• Medicolegal experts take the help of characterization of red cell antigen and antibodies in establishing the
identity of the father in cases of disputed paternities (and also to identity the mother in cases of disputed
maternity). Also, few blood groups provide resistance to some diseases .
 Agglutinogens and
agglutinins
• Agglutinogens refer to the antigens present on the cell membranes of RBCs. A variety of antigens are present on the cell
membrane, but only a few of them are of practical significance.
• Agglutinins refer to the antibodies against the agglutinogens. These are present in the plasma.
• Agglutination of RBCs can be caused by the antigens present on their cell membranes in the presence of suitable
agglutinins (antibodies).
Blood grouping systems:
Depending upon the type of agglutinogen present or absent on the red cell membranes, various blood grouping systems
are known, which can be classified as:
1. Major blood group systems are based on the presence of agglutinogens which are widely prevalent in the population
and are known to cause worst transfusion reactions. These include:
- The classical ABO blood grouping system and
- Rhesus (Rh), (CDE) blood grouping system.
2. Minor blood group systems are based on the presence of agglutinogens which are found only in small proportion of
the population and occasionally produce mild transfusion reactions. These include:
- M and N blood grouping and
- P blood group system
From clinical point of view only major blood group systems, i.e. classical ABO and Rh (CDE) blood grouping systems are
important.
 Landsteiner law
Karl Landsteiner in 1900, framed a law in relation to agglutinogens and agglutinins, which states that:
• If an agglutinogen is present on the red cell membrane of an individual, the corresponding agglutinin
must be absent in the plasma and
• If an agglutinogen is absent from the cell membrane of RBCs of an individual, the corresponding
agglutinin must be present in the plasma.
The Landsteiner law is applicable to ABO blood group system only. The law is not applicable to other
blood group systems because there are no naturally occurring agglutinins in these systems.
Classical ABO blood grouping system:
• The classical ABO blood grouping system is based on the presence of A and B agglutinogens on the
cell membrane of RBCs.
• The A and B antigens present on the membranes of RBCs are also present in many other tissues like
salivary glands, pancreas, kidney, liver, lungs and testis; and also in body fluids like saliva, semen and
amniotic fluid.
• Anti-A agglutinin and anti-B agglutinin refer to the antibody, i.e. which reacts with or acts on the
antigen A and antigen B, respectively.
 TYPES OF ABO BLOOD GROUPS
Depending upon the presence or absence of A and B agglutinogens and α and β agglutinins,
there are four types of blood groups.
1. Blood group A is characterized by:
- Presence of A agglutinogen and absence of B agglutinogen on the cell membrane of RBCs.
- Presence of anti-B agglutinin and absence of anti-A agglutinin from the plasma.
2. Blood group B is characterized by:
- Presence of B agglutinogen and absence of A agglutinogen on the cell membrane of RBCs and
- Presence of anti-A agglutinin and absence of anti-B agglutinin from the plasma.
3. Blood group AB is characterized by:
- Presence of both A and B agglutinogens on the cell membrane of RBCs and
- Absence of both anti-A and anti-B or agglutinins from the plasma.
4. Blood group O is characterized by:
- Absence of both A and B agglutinogens on the cell membrane of RBCs and
- Presence of both anti-A and anti-B agglutinins in the plasma.
 INHERITANCE OF ABO BLOOD GROUPS
• Agglutinogens A and B or the non-antigenic substances which determine the blood groups are
genetically inherited as Mendelian dominant in the classical Mendelian pattern. The ABO phenotypes
and possible genotypes are as under:
Phenotype Genotype
Blood group A AA AO
Blood group B BB BO
Blood group AB AB
Blood group O OO

• Inheritance of classical ABO blood grouping (A, B, AB and O) depend upon three genes, A, B and O
(named after A, B, and O factors). The blood group of offspring depends on two genes which are
inherited from each parent.
• Agglutinogens A and B first appear in the sixth week of fetal life. Their concentration at birth is one-fifth
of adult level and it progressively rises during puberty and adolescence.
• Anti-A (or a) and Anti-B (or b) agglutinins (specific blood group antibodies) are absent at birth, but they
appear 10–15 days after birth and reach a maximum concentration by the age of 10 years.
• The probable mechanism of appearance of α and β agglutinins is described. Antigens very similar to A and B
antigens are commonly present in the intestinal bacteria and foods. When the newborn is exposed to these
antigens, these are absorbed into the blood and stimulate the formation of antibodies against the antigens
recognized as non-self (i.e. not present in the own body) by the immune system.
DETERMINATION OF ABO BLOOD GROUPS
The ABO blood group of an individual can be determined by mixing one drop of suspension of the red cells (in
isotonic saline) with a drop each of antiserum A (containing α agglutinins) and antiserum B (containing β
agglutinins) separately on a glass slide. The antiserum A will cause agglutination (clumping of RBCs having A
antigens) and antiserum B will cause agglutination of RBCs having B antigens). The blood group of the
individual will be shown by the presence of agglutination with one, both or none of the sera.
Note. The antisera A and B are available commercially. For a quick identification, the anti-A serum is tinted
blue and anti-B serum is tinted yellow.
RHESUS (Rh) BLOOD GROUPING SYSTEM
Rh ANTIGENS
• The antigens responsible for this blood grouping system are called Rh antigens or Rh agglutinogens or Rh
factor because these were first discovered in the RBCs of rhesus monkeys. Based on the presence of Rh
antigen, two types of blood groups are described:
- Rh positive blood group and
- Rh negative blood group.
• The Rh antigens were discovered by Landsteiner and Weiner in 1940. They noticed that when RBCs of
rhesus monkey (monkey with red ischial callosity) were injected into a rabbit, antibodies were formed
against these RBCs. When such rabbit’s serum was tested against human red cells, agglutination occurred
in 85% of the cases, i.e. these person’s RBCs contained antigen which reacted with antibodies formed
against rhesus monkey RBCs. They labelled this antigen as Rh antigens and such persons as Rh +ve. The
remaining 15% were labelled as Rh −ve.
• Three types of Rh antigens, viz. C, D and E have been recognized. However, D antigen is commonest and
produces worst transfusion reactions. Therefore, for all practical purposes, the term Rh antigen refers to D
antigen.
• Rh antigens are integral membrane proteins.
Note. Rh agglutinogen has not been detected in other tissues and body fluids like A and B agglutinogens.
Rh ANTIBODIES
There are no natural antibodies of Rh antigens, while in ABO system of blood grouping α or β antibodies are
always present naturally if the appropriate antigen is absent. Rh antibodies (also called anti-D) are produced
only when an Rh −ve individual is transfused with Rh +ve blood or when a Rh −ve mother gives birth to Rh
+ve baby (Rh +ve RBCs of foetus enter into the maternal circulation), Rh antibodies are of IgG type and can
cross the placenta. Since these react best at body temperature so are also called warm antibodies. Once
produced, the Rh antibodies persist in the blood for years and can produce serious reactions during the
second transfusion.
INHERITANCE OF Rh ANTIGENS
• The Rh antigen (D antigen) is inherited as dominant gene D. When gene D is absent from a chromosome, its
place is occupied by the alternate form (allelomorph) called ‘d’. Rh gene is inherited from both the father
and the mother.
• Rh +ve individual may have two genotypes. DD (homozygous) or Dd (heterozygous) of 85% Rh +ve
individuals about 35% have DD genotype and 50% have Dd genotype.
• The genotype of Rh −ve individual is dd.
• Therefore, the genotype (gene composition) of offspring will be:
- DD when gene D is carried by both sperm and ovum
- Dd when one gamete carries D and other d and
- dd, when both the gametes carry gene d.
HAEMOLYTIC DISEASE OF NEWBORN
Haemolytic disease of newborn occurs as a result of incompatibility of Rh blood groups between the mother
and the fetus.
Mechanism of haemolytic disease of newborn in Rh incompatibility
Mechanism of development of haemolytic disease of the newborn can be described under following steps:
1. Entrance of Rh +ve fetal RBCs into Rh −ve mother’s circulation during first pregnancy. At the time of
delivery, the fetal RBCs enter maternal circulation because of severance of umbilical cord. Before delivery,
usually the foetal and maternal circulation do not mix. Since the Rh +ve RBCs enter maternal circulation
during delivery, so the first child is usually normal.
2. Production of Rh antibodies (anti-D) in mother. During postpartum period, i.e. within a month after
delivery, the mother develops Rh antibodies in her blood. As mentioned earlier, the Rh antibodies are of
IgG type and are able to cross the placental barrier. Once formed the Rh antibodies persist for a long
period in mother’s blood.
3. Rh incompatibility reaction during second pregnancy. When the Rh −ve mother in the second pregnancy
also bears a Rh +ve child, the Rh antibodies present in the mother’s blood enter the fetal circulation by
crossing the placental barrier and cause agglutination of fetal RBCs leading to haemolytic disease of
newborn.
Manifestations of haemolytic disease of newborn
Depending upon the severity, the haemolytic disease of newborn may manifest as:
- Erythroblastosis fetalis,
- Icterus gravis neonatorum,
- Kernicterus and
- Hydrops fetalis.
 A B

Hemolytic disease of the new born: (A) Hydrops foetalis; (B) Kernicterus
1. Erythroblastosis fetalis is characterized by:
- Erythroblastosis, i.e. appearance of large number of erythroblasts in the peripheral blood.
- Anaemia occurs due to excessive haemolysis of RBCs by Rh antibodies. Infant may even die of severe
anaemia.
2. Icterus gravis neonatorum
- Jaundice may occur within 24 h of birth due to excessive formation of bilirubin as a result of excessive
haemolysis of RBCs.
- Liver and spleen are enlarged.
3. Kernicterus. It is a neurological syndrome occurring in newborns with severe haemolysis. The excessive
bilirubin formed may enter the brain tissue as the blood–brain barrier is not well developed in infants and
cause damage. The bilirubin mostly affects the basal ganglia producing disturbance of motor activities. It
usually develops when serum bilirubin level exceeds 18 mg/dL.
4. Hydrops fetalis, i.e. the fetus is grossly oedematous. It occurs when haemolysis is very severe. Usually,
there occurs intrauterine death of fetus or if born prematurely or even at term, the infant dies within a
few hours.
Prevention and treatment
Prevention of haemolytic disease of newborn.
The haemolytic disease in the newborn during second pregnancy can be prevented by injecting single dose of
Rh antibodies (anti-D) in the form of Rh-immunoglobulin to mother soon after child birth (1st pregnancy).
These antibodies will destroy the Rh +ve RBCs of the fetus which have gained access to maternal circulation. In
this way active antibodies will not be formed by the mother.
Treatment of haemolytic disease of newborn.
Treatment of haemolytic disease of the newborn is replacement of baby’s Rh +ve blood with Rh −ve blood
exchange transfusion.
CLINICAL APPLICATIONS OF BLOOD GROUPING
1. In blood transfusion. Before blood transfusion always crossmatching is done.
2. In preventing haemolytic disease in newborn due to Rh incompatibility.
3. In paternity disputes. The ABO and Rh blood grouping is helpful in settling cases of disputed paternity.
Antigens A and B are dominant, whereas O is recessive. It is possible to prove that a person could not have
been the father but not that he was or is father; e.g.
- If the child’s blood group is O, whatever the blood group of the mother, a person with blood group AB
cannot be father.
- If the child’s blood group is AB, whatever the blood group of the mother, a person with blood group O
cannot be the father. The predictive value of such a test is strengthened further if several blood group
systems are considered. DNA fingerprinting can prove or disprove fatherhood with 100% certainty.
4. In medicolegal cases. Any red stain on clothing may be claimed to be blood by a supposed victim.
Therefore, it is first confirmed that it really is blood by preparing hemin crystals from the stain extract.
5. In knowing susceptibility to diseases. The incidence of certain diseases is related to blood groups, e.g.
- Individuals with blood group O (non-secretors) are said to be more susceptible to duodenal ulcer (peptic
ulcer).
- Individuals with blood group A are more susceptible to carcinoma of stomach.
BLOOD TRANSFUSION
INDICATIONS
Blood transfusion is a life saving measure and should be carried out when it is absolutely essential. Common
situations in which blood transfusion is indicated are:
6. Blood loss. Severe blood loss is the most important indication for blood transfusion.
7. For quick restoration of haemoglobin in patients with severe anaemia which is required in situations like
pregnancy and emergency surgery.
8. Exchange transfusion is required in haemolytic disease of newborn.
9. Blood diseases like aplastic anaemia, agranulocytosis, leukaemias, haemophilia, purpura and clotting
defects may require blood transfusion.
10. Acute poisoning, e.g. carbon monoxide poisoning.
DONOR AND RECIPIENT
Donor refers to a person who donates the blood and the person who receives blood is a recipient.
Precautions to be taken while selecting a donor are:
• Donor should be healthy and aged between 18 and 60 years.
• Pregnant and lactating mothers preferably should not donate blood.
• Donor should be screened to exclude the diseases which are spread through blood such as AIDS, viral
hepatitis, malaria and syphilis.
• Haemoglobin and packed cell volume (PCV) of the donor should be within normal range. Its approximate
concentration is tested.
Universal donor. Blood of the individuals with blood group O does not contain any agglutinogen. So when this
blood is transfused to a person with any blood group (A, B, AB or O), theoretically its RBCs will not be
agglutinated. Because of this fact, an individual with blood group O is called universal donor. However,
practically this term is no longer valid, as it ignores the complications produced by existence of Rh factor and
other blood group systems.
Universal recipient. Blood of an individual with blood group AB does not contain any agglutinins. So,
theoretically when such an individual receives blood from the individual with any blood group (A, B, AB or O),
there should be no transfusion reaction. Because of this fact an individual with AB blood group is called
universal recipient. However, practically this term is no more valid because it ignores the complications
produced by the existence of Rh factor and other blood group systems.
PRECAUTIONS TO BE OBSERVED DURING BLOOD TRANSFUSION
1. Absolute indication should always be there for the transfusion of blood.
2. Crossmatching should always be done before the blood transfusion. For it blood is collected from donor
as well as recipient. Plasma and RBCs are separated in each. The crossmatching involves two steps: major
and minor crossmatching.
- Major crossmatching involves mixing of donor’s cells with recipient’s plasma. This is called major
crossmatching because of the fact that when mismatched blood is transfused in a recipient, the
donor’s cells get agglutinated as against their agglutinogen there is sufficiently high concentration of
agglutinins in the recipient’s plasma.
- Minor crossmatching involves mixing of recipient’s cells with donor’s plasma. This is called minor
crossmatch due of the fact that reaction of donor’s plasma and recipient’s cells usually does not occur
or is very very mild on giving mismatched blood transfusion because:
- Firstly, the donor’s plasma in the transfusion (about 250 mL) is usually so diluted by the much larger
volume of recipient’s blood (about 5 L) that it rarely causes agglutination even when the titre of
agglutinins against the recipient’s cell is high, and
- Secondly, donor’s agglutinins are also neutralized by soluble agglutinogen which are found free in the
recipient’s body fluid.
3. Donor’s blood should always be screened for diseases which are spread through blood, such as AIDS,
hepatitis B, malaria and syphilis.
4. Rh +ve blood should never be transfused to Rh -ve person. It is particularly must for females at any age
before menopause, because once she is sensitized by the Rh antigen, the anti-D antibodies are formed
and she will not be able to bear a Rh +ve fetus. In other words, Rh +ve transfusion may make a woman
permanently childless.
5. Blood bag/bottle should be checked for the name of recipient and blood group on the label before
starting the blood transfusion.
6. Blood transfusion should be given at slow rate. If rapid transfusion is given, citrate present in stored
blood may cause chelation of calcium ions leading to decreased serum calcium level and tetany.
7. Proper aseptic measures must be taken during transfusion of blood.
8. Careful watch on recipient’s condition is must for the first 10−15 min of starting the transfusion and from
time to time later.
HAZARDS OF BLOOD TRANSFUSION
9. Mismatched transfusion reactions. Mismatched transfusion reaction is the most serious and potentially
fatal hazard of blood transfusion. It is characterized by showing effects of inter-group blood transfusion:
- Agglutination of donor’s red blood cells in the recipient circulation.
- Tissue ischaemia occurs due to blockage of certain vessels by the agglutinated cells. Soon patient
complains of violent pain in back or elsewhere and tightness of chest.
- Haemolysis of agglutinated red cells occurs rapidly releasing large amount of haemoglobin in circulation
(haemoglobinaemia).
 - Haemolytic jaundice may occur due to excessive formation of bilirubin from haemoglobin released by
haemolysed RBCs.
- Renal vasoconstriction is caused by toxic substances released from the haemolyzed RBCs.
- Circulatory shock occurs due to loss of circulating red cells and release of toxic substances leading to fall
in arterial blood pressure and decreased renal blood flow.
- Haemoglobinuria occurs when total free haemoglobin becomes more than that can bind with
haptoglobin (plasma protein binding haemoglobin). The extra free haemoglobin leaks through
glomerular membrane and is passed in urine producing haemoglobinuria.
- Renal tubular damage. If urine is acidic and glomerular filtration is slow, the free haemoglobin passing
through glomeruli is precipitated in the tubules as acid haematin. This obstructs the lumen of tubules
producing renal tubular damage.
- Acute renal shut down (anuria) sets in ultimately due to the combined effects of renal vasoconstriction,
circulatory shock, hypotension and renal tubular damage. Acute renal shut down usually occurs within a
few minutes to few hours after transfusion of mismatched blood and continues.
- Uraemia (increased nitrogenous substances and potassium in the body) results due to acute renal
failure, soon producing coma and death.
2. Circulatory overload due to hypervolaemia may occur following blood transfusion when the transfusion is
rapid specially in patients with cardiac diseases.
3. Transmission of blood-borne infections such as AIDS, viral hepatitis, malaria, syphilis, etc. may occur to
recipient from the infected donor.
4. Pyrogenic reaction characterized by fever
and chills may occur probably due to
destruction of leucocytes and platelets by
antibodies against them.
5. Allergic reactions such as skin rashes and
asthma may occur if donor blood contains
substances to which patient is allergic.
6. Hyperkalaemia may occur after excessive
transfusion because K+ concentration in
stored blood is high. Owing to leakage of K+
from the RBCs into the plasma.
7. Hypocalcaemia producing tetany may occur
following massive transfusion of citrated
blood.
AUTOLOGOUS BLOOD TRANSFUSION
Autologous blood transfusion refers to transfusion of an individual’s own blood which has been withdrawn
and stored. Autologous transfusion is done under the following situations:
For elective surgery, a self-predonation is a common practice in some hospitals.
During surgery, the cell-saver machine when used sucks up the blood from the wound, recycles it and
returns it to the patient’s body.
STORAGE OF BLOOD FOR TRANSFUSION
Some facts about the storage of donated blood are:
• One unit of blood (420 mL) can be collected from a donor at a time under all aseptic measures. An
individual can safely donate one unit of blood every 6 months. Acidcitrate-dextrose (ACD) mixture (120
mL) is added to blood and is stored in sterile container.
• Contents of ACD mixture are:
- Acid citrate (monohydrous), 0.48 g,
- Trisodium citrate, 1.32 g,
- Dextrose 1.47 g and
- Distilled water 100 mL.
• Dextrose (glucose) present in ACD mixture provides energy for maintenance of sodium–potassium pump
activity.
• Anticoagulant activity is provided by the citrates present in the ACD mixture which also decreases the pH of
blood.
• The blood can be stored under above conditions up to 21 days.
• The RBCs in the stored blood swell up due to the following changes as a result of decreased cell metabolism
in cold storage.
- Loss of intracellular K+, which increases plasma K+ concentration from 4–5 mEq/L to 20–30 mEq/L,
- Increase in intracellular Na+ from 12 mEq/L to 30–40 mEq/L
- Increase in intracellular water content. Because of the above changes the RBCs become more
spherocytic and their haemoglobin in hypotonic solution increases. Such cells may rupture in vitro
even in 0.8% NaCl solution. With reference to Na+ and K+ content, volume, shape and saline fragility,
the RBCs become normal within 48 h of transfusion.
• WBCs and platelets in stored blood are virtually absent after 24 h of storage. Therefore, stored blood is not
a suitable medium for transferring WBCs and platelets to a recipient.
• After transfusion of stored blood, 80% RBCs survive for 24 h and thereafter surviving cells are destroyed at
a rate of 1% per day.