IMMUNOHEMATOLOGY &

BLOOD BANKING ,
TRANSFUSION MEDICINES
VIVEK KUMAR RATHORE
PATHOLOGY SECTION INCHARGE
AT DNA LABS
CONTENTS
� INTRODUCTION
� CRITERIA FOR DONATION

� TYPES OF BLOOD GROUP

� SCREENING OF BLOOD

� DETERMINATION OF BLOOD GROUP

� INDIRECT ANTIGLOBULIN (COOMB’S) TEST

� DIRECT ANTIGLOBULIN (COOMB’S) TEST

� COMPATIBILITY TEST (CROSS MATCHING)

INTRODUCTION
� Blood banking is the process that takes place
in the lab to make sure that donated blood, or
blood products, are safe before they are used
in blood transfusions and other medical
procedures.
� Blood banking includes typing the blood for
transfusion and testing for infectious diseases.
� Leukocyte testing for organ
transplantation.
� Laboratory resolution of
parentage problems.
 INTRODUCTION
� The science of Immuno-hematology deals with the
basic principles of antigen and antibody structure, to
understand the principles of compatibility testing and
transfusion reactions the basic knowledge of Immuno-
hematology is essential.
CRITERIA FOR DONATION
To donate blood, you must:

� Be in general good health.

� Not have had any symptoms of infection e.g. sore
throat, cough, runny nose or diarrhoea for at least 1
week.
� Not have had a fever in the last 4 weeks.
� Not have taken medication, herbal supplements or
traditional herbal remedies for at least 3 days. If you
have taken antibiotics, wait at least 1 week.
� Weigh at least 45 kg.
� Have a haemoglobin level of at least 13.0 g/dl for
males and 12.5 g/dl for females.
� It may be harmful to you or the recipients if you
donate blood when you are not eligible to do so.
TYPES OF BLOOD GROUP

� There are more than 30 antigen present on

human RBC.
� There are major four types of Blood Group.
Rh FACTOR
� In addition to antigens of ABO system, the red cells of humans also contain
an additional antigen, called Rh antigen (or Rh factor).

� There are several varieties of Rh antigen—C, D, E, c, d, and e—but the D

antigen is the most common, and antigenically, the most potent. Therefore,
Rh +ve persons are also called D +ve and Rh –ve are called D –ve.

� Persons whose red cells contain this additional antigen are called “Rh
positive” ( Rh +) while those who lack this antigen are called “Rh negative”
(Rh –).

� However, there are no naturally occurring antibodies against Rh (D)

antigen.
� The Rh (D) antigen is not present in body fluids and tissues, but only on
red cells.
Clinical Significance of Rh
factor
� THERE ARE TWO IMPORTANT CLINICAL
SIGNIFICANCE

In transfusions. When an Rh –ve person receives Rh +ve blood,

there is no immediate reaction since there are no antibodies. But
during the next few weeks/months, he/she may produce anti-Rh
antibodies that will remain in the blood. (Even 0.5 ml of Rh +ve
blood is enough to produce immune response). However, if within
a few weeks, or even years later, a second Rh +ve blood is
injected, the newly donated red cells will be agglutinated and
hemolysed, thus resulting in a serious transfusion reaction.
 In pregnancy. The most common problem due to Rh
incompatibility may arise when an Rh –ve mother (phenotype dd)
carries an Rh +ve fetus
� Normally, no direct contact occurs between maternal and fetal
bloods. However, if a small amount of Rh +ve blood leaks (at the
time of delivery) from the fetus through the placenta into the
mother’s blood, the mother’s immune system will start to make
anti- Rh antibodies.
� As a result, some mothers develop high concentration of anti-Rh
antibodies during the period following delivery. Therefore, the
first-born baby will not be affected.
� However, during the second and subsequent pregnancies, the
mother’s anti-Rh antibodies cross the placental membrane into
the fetus where they cause agglutination and hemolysis. The
clinical condition that develops in the fetus is called “hemolytic
disease of the newborn (HDN)’ or “erythroblastosis fetalis”
SCREENING OF BLOOD
A certain set of standard tests are done in the lab
once blood is donated, including, but not limited to,
the following:
� Typing: ABO group (blood type)
� Rh typing (positive or negative antigen)
� Screening for any unexpected red blood cell
antibodies that may cause problems in the recipient
� Screening for current or past infections, including:
◦ Hepatitis viruses B and C
◦ Human immunodeficiency virus (HIV)
◦ Human T-lymphotropic viruses (HTLV) I and II
◦ Syphilis
◦ West Nile virus
◦ Chagas disease
DETERMINATION OF BLOOD
GROUP

� ABO GROUPING WITH ANTISERA (SLIDE

METHOD).
� ABO GROUPING BY TEST TUBE METHOD.

� GEL CARD METHOD

ABO GROUPING WITH ANTISERA
(SLIDE METHOD).
Slide Method: The requirements include Glass
slides, pasteur pipettes, Applicator sticks and
centrifuge.
The reagents are :
� Anti-A sera (Blue color) : Human polyconal or

murine monoclonal.
� Anti-B sera (yellow color):Human polyconal or

murine monoclonal
� Anti-RhD sera (colorless):Human polyconal or

murine monoclonal
� Dangle the hand down to increase the flow of
blood in the fingers
� Clean the fingertip to be pierced with spirit or
70% alcohol (usually ring or middle finger).
� With the help of the sterile lancet, pierce the
fingertip and place one drop of blood in each of
the circular cavities.
� Add one drop of antiserum into each cavity
� Mix each blood drop and the antiserum using a
fresh mixing stick.
� Observe agglutination in the form of fine red
granules within 30 seconds. Anti RhD takes a
slightly longer time to agglutinate compared to
Anti A and Anti B
ABO GROUPING BY TEST TUBE
METHOD
TEST TUBE METHOD : There are two types of
grouping.
� Forword Grouping.

� Reverse Grouping.

Requirements : The requirements includes 6

test tube for both grouping, pasteur pipettes,
5% RBC cell Suspension, Test tube stand,
centrifuge.
The Reagents: Anti-A, B, D for Grouping
Forword Grouping.
� Take 3 Test Tubes and label them with A , B
and O.
� Add 2 Drops of Antisera- A, B and O to each
Test tube.
� Add 5% Cell suspension in each test tube.
� Incubate for 5-10 min, at Room Temp.
� Centrifuge at 1000 rpm for 1 min.
� Observe the agglutination.
Reverse Grouping

� Take 3 Test Tubes and label them with A , B

and O.
� Add 2 Drops of Patients Serum to each Test
tube.
� Add 1 Drop - A group cells in Test tube A.
� Add 1 Drop - B group cells in Test tube B.
� Add 1 Drop - O group cells in Test tube O.
� Shake Gently.
� Incubate for 5-10 min, at Room Temp.
� Centrifuge at 1000 rpm for 1 min.
� Observe the agglutination
5 % Cell Suspension
� Mix 0.05 ml(5 drops) of sedimentation red
cells with 2 ml of normal saline.
� Centrifuge at 1500 rpm for 1 to 2 min and
discard supernatant.
� Repeat it for 3 times.
� Add 4 ml of normal slime to the sedimented
red cells, mix well to get a 5% suspension of
red cells,
GEL CARD METHOD

� Gel technology system developed by Dr.Yves

Lapierre of France,gives more reproducible and
standardied test results.

� This technology utilizes the differential

migration of RBC agglutinates through a small
microtube containing Dextran Acrylamide
gel,size exclusion gel column.
Uses :
For any immunohematolgical tests that has
hemagglutination as its endpoint.
- ABO and Rh typing
- Typing for other blood group system

- Antibody screening and identification

- Compitability testing including cross matching
ADVANTAGES
� Simple ,reliable ,rapid , very sensitive.
� No need to multiple washing of red cell before adding into
Antihuman globulin (AGH) serum. and no need to add
sensitized controlled cell to all negative AGH tests.
� Less specimen volume needed.
� Greater uniformity between repeated tests.
� No variations among technologist in reading and grading
agglutination.
� Provision of centrifuge calibarted to optimal speed for fix
and corrected length of time , reduces potential error during
this phase.
� The cards can be digitally photographed , downloaded as
permanent laboratory data.
DISADVANTAGES

� Special centrfuge to accommodate the microtube

card.
� Special incubators to incubate microtube cards.
� Pipettes to dispose serum and red cell
suspention.
� Expesive
DIRECT COOMBS TEST
INDIRECT COOMBS TEST
THANK YOU
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