MODULE Rhesus Blood Group System

Hematology and Blood

Bank Technique

12
Notes
RHESUS BLOOD GROUP
SYSTEM

12.1 INTRODUCTION
The Rhesus blood group system is the second most important system in
transfusion practice. Rh typing is routinely performed along with ABO grouping
in all donors and recipients.

OBJECTIVES
After reading this lesson, you will be able to:
z describe the nature of Rh antigens and antibodies
z explain the significance of Rh grouping
z discuss the method of Rh grouping

12.2 ANTIGENS OF THE RH SYSTEM

The most significant antigen of the Rh system is D because it is highly antigenic.
An individual is labeled Rh positive or negative depending on the presence or
absence of the D antigen on the surface of the red cells. The D antigen has greater
immunogenicity than all other red cell antigens. In India, 95 percent of the
population is Rh positive and 5 percent is Rh negative.

Besides antigen D, four other antigens of the Rh system have been described:
C,c, E, e. Unlike the ABO antigens the Rh antigens are only present on red
cells.

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Hematology and Blood
Antibodies
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The Rh antibodies are always immune in nature. They are of IgG subtype and
form after exposure to red cells possessing the D antigen. This exposure may
occur through transfusion or during pregnancy.
Rh antibodies are the most important cause of hemolytic disease of new born
and can cross the placenta to cause destruction of fetal red cells. Once formed
their effect persists for many years. Even if their level in the circulation falls, Notes
subsequent exposure to the antigen results in a very rapid secondary response.

Rh genes
There are two homologous genes on the short arm of chromosome 1 which code
for the nonglycosylated polypeptides that express Rh antigenic activity. One
gene is designated RHD and it confers D activity on the red cell. It is present
in D-positive individuals while D-negative individuals have no genetic material
at this site. The D antigen has no allele. D- negative individuals lack RHD .
At the other adjacent locus, is a gene called RHCE. This determines the presence
or absence of C, c, E and e antigens.

INTEXT QUESTIONS 12.1

1. ................... in all donors and recipients are important to perform Rh typing.
2. Rh system’s most significant antigen is ................... because of its high
...................
3. Rh antigens are only located on ...................
4. ................... & ................... are the two homologous genes on the short arm
of chromosome 1 which expresses Rh antigenic activity.
5. RHD determines the ................... on the red cells and RHCE concludes the
presence or absence of ...................

12.3 SIGNIFICANCE OF RH TYPING

Rh antibodies are immune in nature and form after exposure to red cells
containing D antigen. Exposure can occur during pregnancy or after transfusion
of an Rh negative individual with Rh positive blood. More than 80 percent of
D negative individuals who receive a D positive blood transfusion develop Anti-
D. To prevent this, the blood of all recipients and donors is routinely tested for
D antigen to ensure that D negative individuals are identified and receive D

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Hematology and Blood negative blood only. As Anti-D antibodies can cause hemolytic disease of new
Bank Technique
born, Rh negative pregnant women are screened during pregnancy for the
development of unexpected antibodies.

Du phenotype
Du is a weaker variant of D antigen. Du red cells are agglutinated by some anti-
Notes D antisera but not by others. Those which are not agglutinated by anti-D antisera
can be detected by AHG technique.

Importance of detecting Du
1. Though Du is less antigenic than D, these red cells may be destroyed if
transfused to a patient who has anti-D. Hence these donor units must be
labeled as Rh positive and du testing must be done on donors.
2. Du testing is not done routinely on recipients as they are labeled as Rh
negative and transfused with Rh negative blood.
3. If a neonate is Du positive, he can suffer from HDN if the mother has anti-
D.
4. Rh negative mothers of a Du positive neonate must receive RhIg.

Technique of Rh grouping
In Rh grouping, testing is done only for D antigen. Tests for other antibodies are
performed only when specifically indicated.
The methods for Rh typing are
Slide
Tube
Microplate
Gel card

Types of Anti-D antisera

Different types of anti-D antisera are available commercially. These include
(a) Polyclonal human anti-D serum
(b) High protein antisera: This is used for slide grouping. It has macromolecular
additives and gives rapid, reliable results.
(c) Saline reactive antisera: This is prepared from IgG antibodies which are
modified so that they agglutinate antigen positive cells suspended in saline.

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(d) Monoclonal antisera These are of three types: IgM anti-D monoclonal Hematology and Blood
Bank Technique
antisera, blend of IgM and IgG monoclonal antisera and blend of
monoclonal IgM and polyclonal IgG. IgM anti-D antisera are highly
specific and saline reacting. They react equally well at room temperature
and at 37°C. They can be used for slide or tube test. For Du testing a blend
of IgM and IgG monoclonal is used.

Notes

INTEXT QUESTIONS 12.2

1. Exposure of Rh antibodies on red cells containing D antigen may occur
during ................... & ...................
2. Du red cells are agglutinated by ................... and not by others
3. Detection of non agglutinated Du red cells by Anti –D antisera can be done
by ...................
4. ..................., ..................., ................... and ................... are the four methods
of Rh typing.
5. ................... is used for slide grouping.
6. For slide or tube test ................... and for Du testing a blend of ...................
are used.

12.4 SLIDE TECHNIQUE

This can be performed in emergency or outdoor camps but must not be
performed as a routine test.

Material required
1. Glass slides/white tile
2. Monoclonal Antisera A and Antisera B
3. Glass rod for mixing
4. Marker pen
Sample: Blood collected in a plain vial. Sample must be tested within 48hours.
It should be kept in the refrigerator till processed. There should be no evidence
of hemolysis in the sample.

Method
1. Mark one side of the glass slide as A and the other side as B.
2. Put one drop of antisera A on the side marked as A and one drop of antisera
B on the side marked as B.

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Hematology and Blood 3. Add one drop of test blood sample/20% cell suspension to each antisera.
Bank Technique
4. Mix the blood with the reagent using a clean stick. Spread the mixture over
an area of 15mm diameter.
5. Gently rock the slide to and fro and look for agglutination.
6. Record the result.

Notes Interpretation
Agglutination if present indicates a positive result

Advantages
1. Can be used in emergency and blood camps for preliminary grouping.
2. Easy to perform
3. Quick

Disadvantages
1. Not reliable for weak reactions as negative results cannot be checked
microscopically.
2. Serum testing cannot be performed.
3. The test mixture tends to dry fast.
4. Drying causes aggregation of cells which can be interpreted as agglutination.
5. Less sensitive than tube technique.

A B
Agglutinates No agglutination
of reduces
Blood group A

A B

Blood group B

A B

Blood group O

A B

Blood group A-B

Fig. 12.1: ABO grouping by slide technique

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Tube technique Hematology and Blood
Bank Technique
This is the recommended method for grouping. It involves
z cell grouping or forward grouping: testing test red cells with known antisera.
z reverse or serum grouping: testing serum of donor/patient with known cells.
The procedure for cell and serum grouping is described separately but in all
samples both the procedures should be done simultaneously and the results
crosschecked. Notes

Reagents required
1. Monoclonal Antisera-A and Antisera-B. Antisera-A,B is optional.
2. Normal saline
3. Known cells of group A, B, O

Equipment required
1. Centrifuge
2. Glass tubes 12 × 100mm
3. Glass tubes 75 × 10mm

Cell or forward grouping

In this the donor/patient red cell are tested with known antisera.

Method
1. Check that the name and number of the donor/patient on the vial matches
with the form. Write the same donor number on each tube in which grouping
will be performed.
2. Centrifuge the sample to separate the cells and serum.
3. Prepare a 2-5% cell suspension of test red cells in normal saline as follows
z Add the cells to a pre labeled tube (75 × 10mm) filled three fourth
with normal saline.
z Mix and centrifuge at 1000- 2000 rpm for 1-2 minutes. Decant the
supernatant completely.
z Add saline and repeat the procedure till the supernatant is absolutely
clear.
z After three washes, decant the supernatant and to the cell button add
saline by counting the drops to make a 2-5% cell suspension.(10ml
of normal saline and 0.2ml/0.5ml for 2 and 5% respectively).
z Invert gently several times to make an even suspension.

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Hematology and Blood 4. Label three tubes as Anti-A, Anti-B and Anti-A,B.
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5. Add one drop of Anti-A to tube marked A, one drop of Anti-B to tube
marked B and one drop of Anti-A,B to tube marked A,B.
6. Add one drop of 2-5%red cell suspension of donor/patient to each tube and
mix gently. Leave at room temperature for15- 30min or centrifuge at
1000rpm for 1 minute after 5-10min incubation at room temperature.
Notes 7. Resuspend the cell button and check for agglutination. Also look for any
evidence of hemolysis in the supernatant which is read as a positive result.
8. If no agglutination is seen, the contents of the tube must be examined
microscopically.
9. Record the results.
10. An autocontrol (patient’s serum and cells) can be set up in grouping. No
agglutination should be seen in this tube.

Serum grouping
In this the serum of the donor/patient is tested with known cells. The Acells,
Bcells and O cells are obtained by pooling fresh group A, B and O cells from
at least 3 individuals of these known groups and a 5% cell suspension (1ml of
normal saline and 50µl of washed red cells)is prepared in a similar manner as
the cell suspension in cell grouping by washing in saline. The cell suspensions
must be prepared fresh everyday and may be tested using the corresponding
antisera before use. The unit number from which the pooled red cells are
prepared must be entered in the blood grouping register.

Method
1. Label three tubes as A cell, B cell and O cell.
2. Place two drops of the donor/patient serum in each tube.
3. Add one drop of A cells to tube marked A, one drop of B cells to tube marked
B and one drop of O cells to tube marked as O.
4. Mix the contents by gentle shaking and leave undisturbed at room
temperature for 30-60min or centrifuge at 1000rpm for 1minute.
5. Look for agglutination. Also look for any evidence of hemolysis in the
supernatant which is read as a positive result.
6. If no agglutination is seen, the contents of the tube must be examined
microscopically.
7. Record the results immediately.

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Interpretation Hematology and Blood
Bank Technique
Presence of agglutination or hemolysis is a positive result.
A smooth cell suspension after the button is resuspended is a negative result.

Grading agglutination reactions

The reaction result obtained in grouping is graded as follows: Notes
H Hemolysis .This is a positive result
0 No agglutination, only a smooth suspension
1+ Many small clumps, supernatant has free cells
2+ Many small clumps with clear supernatant
3+ 2-3 clumps, no free cells
4+ One big clump, no free cells

Clear
turbid
supernatant
Red cell supernatant
Clump
4+ 3+ 2+ 1+ weak
Fig. 12.2: Grading agglutination reactions for blood grouping by tube technique
P+1

10

12
11
2
3
4
5
6
7
8
9

anti A
anti B
anti AB
anti ----
AC
BC
OC

Fig. 12.3: Blood grouping by microplate technique

Advantages
1. Easy to perform
2. Accurate
3. The cell mixture can be incubated for a long time without drying.

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Hematology and Blood 4. The centrifugation used enhances the reaction and hence even weak
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antigens/antibodies can be detected.
5. Uses smaller quantity of reagents.

Interpretation of result
The results seen in different blood groups are shown below
Notes Table 12.1
Cell grouping Serum grouping
Group Anti-A B AB Ac Bc Oc
A 4+ - 4+ - 2+ -
B - 4+ 4+ 2+ - -
O - - - 2+ 2+ -
AB 4+ 4+ 4+ - - -

Recording results of ABO grouping

Each blood transfusion centre must have a record sheet in which blood grouping
results are recorded.
All reactions must be graded.
Results must be recorded immediately after the test is performed.

Tallying results of forward and reverse grouping

The results of forward and reverse grouping must tally with each other. If a
disparity is noted between the cell and serum grouping, inform the Blood Bank
incharge and follow the necessary instructions on how to resolve it. Do not
release the unit for transfusion till the discrepancy is resolved.

WHAT HAVE YOU LEARNT

z The most significant antigen of the Rh system is D because it is highly
antigenic. An individual is labeled Rh positive or negative depending on
the presence or absence of the D antigen on the surface of the red cells.
The D antigen has greater immunogenicity than all other red cell antigens.
In India, 95percent of the population is Rh positive and 5percent is Rh
negative. Besides antigen D, four other antigens of the Rh system have been

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described: C,c, E, e. Unlike the ABO antigens the Rh antigens are only Hematology and Blood
Bank Technique
present on red cells. The Rh antibodies are always immune in nature and
form after exposure to red cells containing D antigen. Exposure can occur
during pregnancy or after transfusion of an Rh negative individual with Rh
positive blood. More than 80 percent of D negative individuals who receive
a D positive blood transfusion develop Anti-D. To prevent this, the blood
of all recipients and donors is routinely tested for D antigen to ensure that
D negative individuals are identified and receive D negative blood only. As Notes
Anti-D antibodies can cause hemolytic disease of new born, Rh negative
pregnant women are screened during pregnancy for the development of
unexpected antibodies. Rh typing can be done by slide, tube, microplate or
gel technique.

TERMINAL QUESTIONS
1. Explain the significance of Rh typing
2. Explain the different types of anti-D antisera

ANSWERS TO INTEXT QUESTIONS

12.1
1. ABO grouping
2. D & Antigenicity
3. Red cells
4. RHD & RHCE
5. D activity & C,c,E and e antigens

12.2
1. Pregnancy, transfusion
2. Anti –D antisera
3. AHG technique
4. Slide Tube Microplate & Gel card
5. High protein antisera
6. IgM anti-D antisera, IgM and IgG monocolonal

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