B loo d

G r o p i n
g Dr. Manjiri Makde
Asst. Prof.
Dept of Pathology
GMCH, Nagpur
B L O O D GROUP
SYSTEMS
• The blood group systems are formed by the Antigens present on the RBC’s

• These antigens are sugars/oligosaccharides attached to RBC’s.

• Every individual inherits

a gene  which codes for a
specific enzyme  which
leads to formation of specific
antigen  added on to
RBC

• The type of antigen coded

by the gene determines
the blood group
• Blood group genes are inherited in a Mendelian manner and are mostly
located on Autosomes
• The ABO gene resides on chromosome 9 and Rh gene on Chromosome
1
• Nearly 30 blood group systems are recognized by
ISBT (International Society of Blood Transfusion).
• In blood transfusion practice, most important blood group systems are ABO
and Rh.

• This is because: A, B & Rh D antigens are most immunogenic

• They are capable of eliciting a strong antibody response on stimulation

• Alloantibodies to A, B and Rh D antigens can cause destruction of transfused

RBCs or can induce HDN (hemolytic disease of newborn)

• Next to A, B & D antigens, other antigens important in transfusion medicine

due to their immunogenicity are K, C, Fya, c, E, k, e, Jka, S, and s.
 ABO Blood Group
System
Six main types of blood group
• A (A1 & A2)
•B
• AB (A1B / A2B)
•O

Identification of these 6 blood groups is based on presence or absence

of A and/or B antigen on RBCs
• Antigens of the ABO system : A, B, & H

H antigen
• In addition to RBCs, these antigens are also expressed on WBCs,
Platelets, and various other body tissues & body secretions
 H antigen/ H substance

• H Gene (present on 19th chromosome)  Codes for 1,2 fucosyl transferase enzyme
 converts precursor substance (present on RBCs) into H substance.

• A & B genes  produce specific transferase enzyme  which converts H substance

into A & B antigens resp. (however, some amt. of H antigen remains unchanged on
RBCs)

• The O gene  produces inactive transferase  so the H antigen persists

unchanged on RBCs

• Thus, H substance acts as a precursor to each of ABO blood group antigens

• It is present in all people (except those with Bombay Blood phenotype)

F o r m a t i o n of a n t i g e n s of A B O s y s t e m
 B om b a y B lood G r o p
• Some individuals do not inherit H gene (genotype hh)
• Thus, cannot synthesize H substance  H antigen not formed

• Such persons may inherit A & B gene  but they cannot express it (ie. A, B & H
antigens on RBCs are not formed, as H substance is not produced)

• Such individuals are said to have Bombay Blood Group

• Their RBCs type as Blood group O (phenotype Oh)

• But, as they have no H, A & B antigens on RBCs  their plasma contains strong
anti-H along with anti-A & anti-B antibodies
• Bombay blood group (Oh) persons should be transfused with Oh blood only.
 Landsteiner’s Law

1. If an antigen is present on the RBCs of an individual, the corresponding

antibody must be absent from the plasma
• Applicable to ABO and all other blood groups.

2. If an antigen is absent on the RBCs of an individual, the corresponding

antibody must be present in the plasma
• Applicable to ABO blood group only.
• does not hold good for Rh factor (ie. If Rh factor absent  anti-D absent)
Landsteiner’s Law (cont…..)

H antigen Nil
 Rh blood group system
 Antigens: The important antigens of the Rh system are C, D, E, c, and e.
• D antigen is the most immunogenic.

• Rh antigens are expressed only on red cells and not on any other tissues &
also not secreted in body fluids.

• In contrast to ABO antigens, Rh antigens are fully expressed on RBCs before

birth and also on RBCs of early foetuses.

• Depending on the presence or absence of antigen D on RBCs, a person is

grouped either as Rh positive (when RBCs express antigen D) or Rh negative
(when D antigen is absent on RBCs).
 Antibodies:
• Rh antibodies are of immune type, i.e. they are the result of immunization by
blood transfusion or pregnancy.

• Most of these antibodies are of IgG class.

• These Rh antibodies can cause haemolytic transfusion reaction or haemolytic

disease of newborn.

• Due to high immunogenicity of D antigen, Rh-negative persons (especially

women of child bearing age) should be transfused only with Rh-negative
blood.
• During pregnancy, IgG anti-D can cross the placenta and induce
haemolytic disease of newborn by causing immune haemolysis of foetal
red cells.

• Rh haemolytic disease of newborn can be prevented by prophylactic

administration of Rh immune globulin to all Rh-negative women during
mid pregnancy and within 72 hours of delivery.

• Anti-D and anti-c can cause severe HDN.

• Anti-C, anti-E, and anti-e usually cause mild HDN
Serologic Tests For Blood
Grouping
 ABO Grouping

 2 methods
• Cell grouping (forward grouping)
• Serum grouping (reverse grouping)

Both cell & serum grouping should be done  reduces risk of error
in blood grouping
Cell Grouping / Forward
Grouping
RBCs are tested for presence of A & B antigens by employing known
specific anti-A & anti-B sera.

 5 methods
• Slide test
• Tube method
• Microplate method
• Column agglutination or gel technology
• Solid phase adherence technology
 S l i d e Test

Principle: Red cells from the patient’s sample are tested for A and/or B antigens
by using known reagent antisera (anti-A and anti-B).
• Agglutination of red cells indicates presence of corresponding antigen on
red cells.
Specimen: may be either capillary blood from finger prick, or venous blood
collected in EDTA anticoagulant.

 Reagents:

• Antisera: used to detect antigens on red cells.

• Previously, polyclonal (anti-A, anti-B, and anti-AB) sera, obtained from

humans, were used for cell grouping.

• These now replaced by anti-A and anti-B monoclonal antisera. (Monoclonal

antiserum contains specific antibodies against a specific antigenic epitope
and is produced by a single clone of plasma cells).
• Monoclonal antisera are specific, avid, sensitive, and can detect
weak antigens.

• Anti-A is colour coded blue

• anti-B is colour coded yellow.

• anti-A, B is colourless.
 Method:
1. Divide a clean and dry glass slide into two sections with a glass
marking pencil. Label the sections as anti-A and anti-B .

2. Place one drop of anti-A and one drop of anti-B antiserum in the center
of the corresponding section of the slide.

3. Add one drop of blood sample to be tested to each drop of antiserum.

4. Mix antiserum and blood by using a separate stick for each section.

5. observe for agglutination after exactly two minutes.

 Results:
Positive : small clumps of red cells are seen floating in a clear liquid.

Negative : Uniform suspension of red cells.

Blood group B
Blood group A

Blood group AB Blood group O

 Interpretation:

agglutination

 Uses of Slide test

used as a preliminary grouping test before:
1) blood donation,
2) In blood donation camps, and
3) In case of an emergency.
 Advantages of Slide test: rapid and simple.

 Disadvantages of slide test :

• Weakly reactive antigens (like A1) may be missed by slide method.
• Slide test is also not suitable for serum grouping.
• Drying of the antiserum-blood mixture around the edges can
be misinterpreted as agglutination.
• If the result is read before two minutes, weak antigens may be missed.

Results of slide test should always be confirmed by cell and serum grouping
by tube method.
 T be m e t h o d
 For cell grouping/forward blood grouping:
• known antiserum and patient’s RBCs are mixed in a test tube,

• incubated at room temperature for 5 minutes,

• and then centrifuged.

• Following centrifugation, a red cell button (sediment) will be seen at

the bottom of the tube.

• Cell button is resuspended by gently tapping the base of the tube.

 Results:
• Positive test: Clumps of red cells suspended in a clear fluid
• Negative test: Uniform suspension of red cells.
Separate tubes of : auto control, positive control, and negative control should always be
set up along with the test sample tube.
1. Auto control tube:
• necessary to rule out auto agglutination of patient’s red cells, which occurs
when autoantibodies are present in patient’s serum.
• In auto control tube, patient’s red cells are mixed with patient’s own serum and the tube is
centrifuged;
• if agglutination occurs, it is autoantibody-induced.
• Auto control test is particularly essential when ABO grouping is being done only
by forward method and blood group is typed as AB.

2. In two positive control tubes,

• anti-A serum is mixed with group A red cells and
• anti-B is mixed with group B red cells, respectively.
3. In two negative control tubes,
• anti-A serum is mixed with group B red cells and
• anti-B serum is mixed with group A red cells, respectively.

• These controls are necessary to confirm that reagents are working

properly.

 Advantages of tube method:

• more reliable than slide method  as centrifugation brings antigen
and antibodies closer together & allows detection of weaker antigen
antibody reactions.
 Microplate method
• This technique is commonly used in many blood bank laboratories.
• A microwell plate consists of 96 small wells (U- or V- shaped or flat-
bottom shaped).
• U-type is preferable since results are easier to read.

• Advantages: This method is

• more cost-effective since very less antisera is required as compared to
the test tube method;
• also multiple samples can be tested at the same time because of
96 wells.
Serum grouping/ Reverse Grouping
• Serum is tested for presence of anti-A & anti-B antibodies by
employing known group A & group B reagent RBCs

• Not carried out in infants below 4 months : as they start producing

anti-A & anti-B antibodies by 4-6 months of age

• In serum or reverse grouping, serum from the patient and reagent

red cells of known group (available commercially or prepared in the
laboratory) are used.
 R h D G r o p in g
• Out of the various antigens of the Rh system, D antigen is the
most immunogenic and therefore, red cells are routinely tested for D.

• Individuals with D antigen on their RBCs are called as Rh-positive,

• While those without D antigen on their RBCs are called as Rh-negative.

• Anti-D antibodies are not naturally occurring  develop only
after exposure to Rh D positive RBCs ie.
* following transfusion of Rh positive blood to Rh
negative individual or
* in mother during pregnancy -- where mother is
Rh
negative and Fetus is Rh positive ).

• If Rh-negative individuals  transfused with Rh-positive blood  90% of

them will produce anti-D antibodies.

• In such sensitised individuals  re-exposure to D antigen can cause 

haemolytic transfusion reaction or, in pregnant women  haemolytic
•Rh D grouping is done only by forward/cell grouping
method.

•Serum/reverse grouping is not carried out because of

the absence of anti-D in Rh-negative persons.

•Method of Rh D grouping is similar in principle to ABO

grouping.

•Since serum or reverse grouping is not possible, each

sample is tested in duplicate in blood banks.

•Autocontrol (patient’s red cells + patient’s serum) and

positive and negative controls are included in every test
Blood group: ‘A’ Rh Positive
run.
• Blood units positive with both D and weak D are labelled as  Rh D-positive.

• Blood units negative with both D and weak D are labelled as  Rh-negative.

• Monoclonal IgM anti-D antiserum should be used for cell grouping 

allows Rh grouping to be carried out at the same time as ABO grouping at
room temperature.

• With monoclonal antisera, most weak forms of D antigen are detected and
further testing for weak forms of D antigen (Du) is not required.
 C O M PAT I B I L I T Y
TEST/CROSS M AT C H
These are a set of procedures  carried out before issuing the blood
to patient for transfusion.
These steps include:

1. Checking of
• patient’s blood requisition form
• blood bank records
- to know the reason for transfusion,
- history of previous transfusions,
- result of previous blood grouping test,
 - to know any clinically significant antibodies detected,
- any significant adverse effects to transfusion.

2. Performing ABO and Rh grouping of patient’s blood sample

and checking against previous results.

3. Confirmation of ABO and Rh blood group of donor unit

4. Antibody screening and identification

 5. Cross matching / Compatibility testing :
• Consists of :
-testing patient’s serum against donor RBCs  to detect any antibodies in
patient’s serum, reacting with donor RBCs (major cross match)

-testing patient’s RBCs against donor serum  to detect any

donor antibodies reacting with patient’s RBCs (minor cross match).

• Donor blood is considered to be compatible if there is no agglutination

or haemolysis in major or minor cross match.
 Cross matching is performed to:

• Confirm compatibility between ABO blood groups of the donor and the
recipient (by major and minor cross match tests)

• Detect any irregular antibodies present in recipient’s serum

reactive against donor’s RBCs (by antibody screening).
-Those antibodies other than anti-A and anti-B in recipient’s serum are
called as irregular or unexpected antibodies.
- They may be naturally occurring or immune.
- These antibodies in recipient's serum are capable of
 destroying transfused Donor’s RBCs (carrying the
relevant antigens like D, C, c, E, e, K, k, Fya, Fyb).
• Minor cross match is considered as less important  as antibodies in
donor blood unit get diluted or neutralised in recipient’s plasma.

6. Labelling of blood product with

- identifying information of the recipient,
-compatibility/cross match test result
before issuing the blood for blood transfusion.
 TESTS/ METHODS OF CROSS MATCHING:
• A single tube cross match consisting of three stages is recommended.

• Recipient’s serum is tested against donor’s RBCs under three

different conditions;
• Agglutination or haemolysis in any one of the three stages
indicates incompatibility.

• The three stages of compatibility test are as follows:

1. Compatibility test at room temperature
2. Compatibility test at 37° C
3. Indirect antiglobulin test (Indirect Coomb’s Test)
1. Compatibility test at room temperature spin cross
(Immediate match):
• The purpose of this test is to detect ABO incompatibility.

• Saline-suspended donor’s RBCs and recipient’s serum are mixed

in a test tube, incubated briefly at room
temperature, and centrifuged.

• Agglutination or haemolysis indicates incompatibility.

• If agglutination or haemolysis is absent, next step is performed.

2. Compatibility test at 37°C:

• The test tube from test performed at room temp.

( ie. Test tube from stage 1) is:
- incubated at 37° C for 20 minutes,
- centrifuged again, and
- examined for agglutination.

•Agglutination or haemolysis indicates

incompatibility

• Ifagglutination is absent, perform the last stage (ie.

stage 3).
3. Indirect antiglobulin test (Indirect Coomb’s Test):
• The mixture from stage 2 is:
- incubated at 37° C for 30-60 minutes,
- washed in saline,
- antiglobulin reagent (Coomb’s sera) is added.
- re-centrifugation,
- examine for agglutination or haemolysis.
• This test detects most of the clinically significant IgG antibodies.
• If agglutination or haemolysis is not observed in any of the above stages,
donor unit is considered to be compatible with recipient’s serum.
MICROBIOLOGICAL SCREENING
OF BLOOD
• HIV BY ELISA
• HBV BY ELISA
• HCV BY ELISA
• VDRL FOR SYPHILIS
• MALARIA (PERIPHERAL SMEAR)
B L O O D COMPONENTS
and DERIVATIVES
W H O L E BLOOD

INDICATIONS:
• ACUTE BLOOD LOSS
• EXCHANGE TRANSFUSION IN NEONATES
• NON AVAILABILITY OF PRC

CONTRAINDICATIONS
• CHRONIC ANEMIA
PRC

INDICATIONS:
• CHRONIC SEVERE ANEMIA
• ANEMIA WITH CCF
• ACUTE BLOOD LOSS
PLATELETS

INDICATIONS:
• THROMBOCYTOPENIA
• PLATELETS FUNCTION DISORDER
• MASSIVE BLD TRANSFUSION

CONTRAINDICATION
• TTP
• HUS
FFP

INDICATIONS
• DIC
• LIVER DISEASE
• TTP
• WARFARIN OVERDOSE
• MASSIVE BLD TRANSFUSION
CR Y O P R E C I P I T A T E

INDICATIONS
• hemophilia
• vWb disease
• Deficiency of fibrinogen
Collection and Storage of
B loo
Donor
Collection of blood  it is a process whereby One unit (350ml) whole
dblood is collected from a suitable donor in an anticoagulant solution.
Proper selection of blood donors is necessary  as all
blood components & blood products are obtained from the Donor
blood.

 Types of blood donors:

• Voluntary donors
• Replacement donors
• Professional donors
 Criteria for Selection of blood donors:
Donor selection process consists of four parts:
• Predonation counselling
• Medical history
• Physical examination
• Haemoglobin estimation

 Predonation Counselling
• This is an education programme in which  information about health
condition, high-risk behaviour, self-exclusion/self-deferral, and
procedures involved in blood donation is given, donor’s questions are
answered, informed consent is obtained, and reassurance is given in
case of anxiety.
 Medical History
• Age: The lower and higher age limits for blood donation are  18 and 60
years, respectively.

• Donation Interval : The interval between two consecutive blood

donations should be at least 3 months  to avoid iron depletion in the
donor.

• Volume of Donation : An individual weighing 45 kg or more can safely

donate blood up to 350 ml.

• Pregnancy and Lactation : Pregnant women and lactating mothers (up to

1 year post-partum) should not donate blood.
• Infectious Diseases :
Blood should not be collected from donors :
- who give history suggestive of HIV infection

- individuals exposed to the risk of HIV infection (homosexuals;

intravenous drug abusers; or individuals having contact with commercial
sex workers, with multiple sexual partners)

- individuals with history of jaundice within last 1 year or a positive test

for HBsAg or anti-HCV antibodies

- H/O Malarial Infection in last 3 months

- Individuals with diabetes mellitus, hypertension, heart disease, renal
disease, liver disease, lung disease, cancer, epilepsy, bleeding disorder, or
allergic disease

• Drugs: donors taking drugs (e.g. aspirin or other non-steroidal inflammatory

drugs (which affect platelet function), and drugs with teratogenic action like
finasteride, isotretinoin, acitretin, and etretinate, or cytotoxic drugs (like
cyclophosphamide

• Blood Transfusion : A person should not be accepted as blood donor for 6

months after receiving blood transfusion.
• Immunisation:
Nature of vaccine received and respective deferral period are as follows:
- Attenuated live virus vaccine for measles, mumps, yellow fever, Sabin
polio: 2 weeks
- German measles: 4 weeks
- Rabies: 1 year
- Passive immunisation with animal sera: 4 weeks
- Hepatitis B immune globulin: 1 year.

• Skin Piercing : Donors with history of tattooing, electrolysis, ear piercing,

or acupuncture during last 12 months should not be accepted.
 Physical Examination
• Weight: should be minimum 45 kg
•Blood pressure: Systolic blood pressure should be 100 to 180 mm of Hg
and diastolic 50 to 100 mm of Hg
• Pulse: should be 50 to 100/min and regular
• Temperature: should be normal.
• Donor should be in good general health.
• Inspect donor’s arms for possible evidence of intravenous drug
abuse such as scars or infection at venepuncture site.

• Clinical examination of cardiovascular system, respiratory system,

and abdomen should be normal.
 Haemoglobin Estimation
• Screening of donors (for anaemia) in blood bank is done by copper sulphate
specific gravity method.
• A drop of blood is allowed to fall in copper sulphate solution of specific
gravity 1.053 from a height of 1 cm.
• Specific gravity of 1.053 is equivalent to haemoglobin concentration of 12.5
g/dl.
• The drop of blood gets covered with copper proteinate and remains discrete
for 15 to 20 seconds.
• If the blood drop sinks within this time, its specific gravity is higher than that
of copper sulphate solution (i.e. haemoglobin is >12.5 g/dl) and
haemoglobin level is acceptable for donation.
• If the blood drop floats, haemoglobin level is < 12.5g/dl & is unacceptable.
 COLLECTION OF DONOR BLOOD
Blood bag: Blood from a donor is collected in a sterile, disposable
plastic blood bag (single, double, triple, or quadruple bag system) with
capacity to hold 350 ml of blood.
• These bags contain a standard amount of anticoagulant-preservative
solution for specified amount of blood.
 Anticoagulant-preservative solution:

The solution in the blood bag usually contains:

• citrate phosphate dextrose adenine (CPDA)-1 (49 ml for 350 ml of blood).
• This solution  prevents clotting of blood and also provides nutrients
to maintain metabolism and viability of red cells.
• In CPDA-1  blood can be kept stored at 2 to 6°C for maximum of 35 days.

Function of each component of this solution is as follows:

• Citrate: Anticoagulation by binding of calcium in plasma
• Phosphate: Acts as a buffer to minimise the effects of decreasing pH in blood
• Dextrose: Maintenance of red cell membrane and metabolism
• Adenine: Generation of ATP (energy source).
 PROCESSING OF DONOR BLOOD
• This refers to various tests and procedures carried out on the donor
blood after collection but prior to cross matching.
 STORAGE OF DONOR BLOOD
• Whole blood is stored at a temperature between 2° to 6°C in a refrigerator
specifically designed for blood storage.
•At temperatures < 2°C  freezing and lysis of red cells will occur;
transfusion of such haemolysed blood will lead to DIC & acute renal failure in
the recipient

•Proliferation of any contaminating bacteria, which may have gained entry

during venepuncture, may occur at temp > 6° C,

•Glycolysis in red blood cells is kept at a minimum level in between 2° to 6°C

temperature range  so that dextrose in anticoagulant-preservative solution
is not utilised rapidly.
Adverse effects of Transfusion
A d v e r se effects of T r a n s f sio n
 Blood transfusion is a life saving but hazardous procedure

 Benefits of blood transfusion include:

• Improvement of oxygen carrying capacity
• Replacement of coagulation factors, platelets, Immunoglobulins

 It should be considered only when no alternative means is available

• Intravenous fluids (saline, dextrose) should be used when
only volume replacement is required
I m m e d i a t e compl i cat ions of blood
t r a n s f sion
D e l a y e d compl i cat i ons
Tr a n s m i s s i o n of infectio  s organisms
• Questionnaire
• Q.1) Bombay Phenotype (Oh) individuals may have all the following
antibodies, Except:
a) anti-A
b) anti-B
c) anti-H
d) anti-O
e) anti- A,B
• Q.2) All are immediate complications after blood transfusion, Except:
a) Anaphylactic reactions
b) GVHD
c) Febrile reactions
d) Allergic reactions

• Q.3)
• Q.4) Indications for transfusing FFP are all, Except:
a) TTP
b) Acute blood loss
c) DIC
d) Liver Disease

• Q.5) Presence of which antigen on RBCs acts as a receptor for P. Vivax malarial
Infection?
a) Duffy antigens
b) MNS antigens
c) Kidd antigens
d) Kell antigens
e) Lutheran antigens
• Q.6) Difference between immune anti-A & anti-B alloantibodies and
non-red cell stimulated (ie. Naturally occurring ) anti-A & anti-B
alloantibodies is that immune antibodies are :
(True statement about immune antibodies is?)

a) They are generally IgG rather than IgM

b) They are unable to cross placenta
c) They can be enhanced in reactivity by incubation at 4 degree
Celsius
d) They can cause direct agglutination at room temperature
e) They can rarely cause clinical hemolysis
• Q.7) Identify the technique of blood
grouping?
• Q.8) Which of the following statements is False regarding Hemolytic
Disease of Newborn (HDN) caused by Rh antibodies?

a) Fetal hemolysis is typically severe

b) It rarely occurs during first pregnancy
c) It is most common with O mothers & A babies
d) It is most common with Rh negative mothers and Rh positive
babies
• Q.9) Anticoagulant-preservative solution in blood bag contains?

?
• Q.10) In Reverse blood grouping method:

a) Serum is tested for presence of anti-A & anti-B antibodies

b) RBCs are tested for presence of A & B antigens
c) Serum is tested for presence of A & B antigens
d) RBCs are tested for presence of anti-A & anti-B antibodies
Practical combined with SDL 10
INSTRUCTIONS

All students should prepare voice over PPT for following questions and send to the respective practical
batch group. This SDL is compulsory.

1. Name various methods of blood grouping

2. Importance of reverse grouping
3. Draw a well labelled diagram of O negative and B positive blood group.
( Draw on a paper, scan or take a pic and insert)
4. Principal of compatibility testing
5. Importance of Bombay blood group
Thank you