University of Diyala/ College of Medicine
Department of Physiology
Physiology Lab
Blood Grouping and Cross Matching
Dr. Muqdad Abdulameer Younis & Dr. Asmaa Abbas Ajwad
11th January 1
2020
Outlines
• Objectives of blood grouping experiment
• Introduction, Mechanism, and principle
• Methods for determining blood group
• Procedure
• Some clinical implications
Objective
s
To determine the major blood groups (ABO system)
and Rh type of the individual.
To learn the mechanism and principle of
the experiment .
To get an idea about ABO & Rh incompatibility.
Introduction
The surfaces of the RBCs contain numerous glycoprotein markers known as
antigens ( Ag ) . There are many different types of antigens , but the most
common are the A,B, & D antigens ( D is also known as the Rh factor).
According to the presence of these antigens , human blood can be classified into
many groups.
About 6 months after birth , lymphocytes begin to produce certain antibodies
( Ab
) which are proteins that circulate in the blood plasma . These Ab are
isolated from the plasma & used as anti-sera in the laboratory to determine blood
types .
Introduction
Blood grouping : also called blood typing, is a test performed to
define the individual blood group and it is performed by mixing
anti-ABO and anti-Rh agglutinins (antibodies, anti-sera) with the
RBCs .
The erythrocytes of a person contain blood group antigens on the
surface of the membrane. When these antigens are allowed to treat
with corresponding antibodies (agglutinins), antigen-antibody
reaction occurs and form agglutination.
Agglutination
Introduction
There are two important blood group systems :
1. ABO system ( the most important blood group system in blood
transfusion )
2. Rh system (the most important blood group system after ABO )
The difference between ABO and Rh system is that in ABO , the agglutinins
are formed spontaneously and can cause immediate reaction . While in Rh
system ,a spontaneous formation of agglutinins does not occur which can
cause only delayed reactions.
Introduction: ABO
the presence System
ABO blood grouping system divides blood into 4 groups (A,B,AB,O) based on
or absence of antigens A and B on the RBC surfaces and naturally
occurring antibodies anti-A and anti-B on serum. In normal human blood, a
reciprocal relationship exists between antigens and antibodies as described
below:
Introduction : Rh
System
• There are 6 common types of Rh antigens these
are C,D,E,c,d,e .
• The most common is the D antigen . Any body that has D
agglutinogens is Rh positive .
• The antibodies to Rh antigens do not occur naturally but
can occur after blood transfusion and during
pregnancy .
• The percentage of people who are Rh+ is 75 – 85 % .
ABO and Rh Systems
(Summary)
The underlying table shows the relation between blood group,
the
presence of agglutinogens and agglutinins, and their frequency.
Blood type Antigens Antibodies Frequency
O ------------------- Anti-A, Anti-B 46%
A A Anti-B 42%
B B Anti-A 9%
AB A,B ------------------- 3%
Rh + D-antigen
Rh - None
Mechanism and
Principle
Blood grouping is done on the basis of
agglutination .
Agglutination means the collection of separate particles
like RBCs into clumps or masses. Agglutination occurs
if an antigen is mixed with its corresponding antibody .
For example, when A antigen is mixed with anti-A
serum, the anti-A bodies in the serum stick to the A
antigens on the erythrocytes. This Ag-Ab reaction will
give the slide a beaded appearance.
Methods
Slide Method
• Requires less blood and less time to get the result.
• It is the method that we are going to use in today’s lab.
Tube Method (Gold standard method)
• Allows longer incubation of Ag & Ab without drying.
• Tubes can be centrifuged to enhance the reaction.
• Can detect weaker antigen – antibody reaction.
Cross Matching
• This is a direct test of compatibility of donor’s cells
and recipient’s serum . In this test the donors RBCs
are mixed with the recipient serum on a slide . If
agglutination occurs this means that the donor’s blood
is incompatible with the recipient blood .
• It is a prerequisite for blood transfusion to
avoid
reactions of mismatched transfusion .
Materials and Instruments
Clean dry microscope slide.
Antisera ( anti-A serum, anti-B serum, and anti-D
serum)
Capillary blood.
Toothpick for mixing.
Lancet , alcohol 70%, cotton
Wax pencil.
Procedure
1. On a prepared microscope slide with three circles drawn with wax pencil and
labeled A , B , and Rh, add anti-A serum to circle labeled A , Anti-B serum to
the one labeled B , and anti-D ( or anti-Rh ) to circle labeled D . To make it
easier and avoid any confusion, we will use two slides. On one slide put anti-
A serum & anti-B serum and on the other slide put anti-D serum .
2. Place a small drop of blood in each circle ( make sure you have enough blood ,
but do not completely fill the circle … if you do , you have too much blood on
your slide ) .
3. Mix the contents of each circle with a clean toothpick . The reaction will appear
in 20-60 sec.
4. Check for agglutination by naked eye or by microscope and the test should be
performed in a warm room temperature to prevent the action of cold agglutinins
which may cause a confusion . You can place slide on a white piece of paper to
better view the agglutination process .
After mixing ani-sera with the blood,
you will get something as in the
below slides (depending on the blood
type)
Step 2
Pictures
Procedure Steps in
Step 1
Important Note
Be sure to have a clean microscope slide (slides) with
three circles drawn in wax pencil and the circles
labeled A ,B , and Rh ( or D ) available before you
stick yourself with lancet !
How to Read Your
Results
How to Read Your
Results
Universal Recipient Vs Universal
Donor • Plasma of AB group has no antibody. This does not cause
agglutination of RBCs from any other blood group. People with
AB group can receive blood from persons with any blood
Universal group
• Thus, people with this blood group are called universal
Recipient recipients .
• RBC of O group has no antigen ( no Ag-A/ Ag-B ) and
so agglutination does not occur with any other blood group .
• Thus, “O “ group blood can be given to any blood group
Universal persons and people with this blood group are called universal
Donor donors .
Source of
error as well.
Errors
•Inadequate cleaning of the microscope slide. Wet slide can cause an
• Circles not labeled properly.
• Very low room temperature .
• Drying of reaction giving a false positive results .
• Large drops of blood will lead to overflow the circle to other one.
Advantages and Disadvantages of Slide
Method
Advantages Disadvantages
Small sample volume Less sensitive
Fast method Drying of reaction giving to
Preliminary typing test false positive results
Easy
Clinical Implications: Hemolytic
Disease of Newborn
During pregnancy for the Rh+ offspring of Rh-incompatible parents,
when the mother is Rh- and the father is Rh+. The first child of such parents
is usually in no danger unless the mother has acquired anti-Rh antibodies by
virtue of incompatible blood transfusion.
During delivery, however, a small amount of the fetus’s blood may enter the
mother’s bloodstream. The mother will then produce anti-Rh antibodies,
which will attack any Rh-incompatible fetus in subsequent pregnancies. This
process produces erythroblastosis fetalis, or hemolytic disease of the
newborn, which can be fatal to the fetus or to the infant shortly after the
birth.
The disease can be avoided by vaccinating the mother with
Rh immunoglobulin after delivery of her firstborn if there is Rh-
incompatibility. The Rh vaccine destroys any fetal blood cells before the
mother’s immune system can develop antibodies.
Clinical Implications: Hemolytic
Disease of Newborn
Clinical Implications:
Transfusion
blood . The reactions may Reaction
Transfusion Reaction occurs due to transfusion of incompatible
be mild causing only fever & hives
( skin lesions characterized by itching ) or may be severe leading to
renal failure , shock and death.
Clinical Implications: Kidney
• Failure
Because the arms of the Y-shaped antibodies attach randomly to
more than one nonself erythrocyte surface, they form clumps of
erythrocytes. This process is called agglutination.
• The clumps of erythrocytes block small blood vessels throughout
the body, depriving tissues of oxygen and nutrients.
• Asthe erythrocyte clumps are degraded, in a process
called hemolysis, their hemoglobin is released into the
bloodstream. This hemoglobin travels to the kidneys, which are
responsible for filtration of the blood. However, the load of
hemoglobin released can easily overwhelm the kidney’s capacity
to clear it, and the patient can quickly develop kidney failure.
Summary of all the results you may
get:
Take Home Assignment #1: Fill
the Blank Rectangular
Spaces
Take Home Assignment #2:
Case Study
Ahmed is a 17-year-old who was admitted to hospital following a car
crash. He suffered a crushing injury to his right leg. He is booked for
surgery and his blood group is Type AB. Who in your group can give
blood to Ahmed ? Explain briefly?
Thank you
