symptoms of hemolytic transfusion reaction when
transfused with her husband’s group O blood. They noted
that the responsible antibody developed in the mother
through an antigenic factor from the fetus. The antibody
In 1940, Landsteiner and Wiener immunized rabbits
and guinea pigs with red cells of rhesus monkeys. The
serum of immunized rabbits contained an antibody named
anti-Rh which agglutinated red cells in approximately
85% of white population tested. Its antigenic determinant
was called Rh factor. The antibody discovered by Levine
and Stetson in the mother was subsequently reexamined
and found identical in activity as the anti-Rh antibody of
Landsteiner and Wiener. This work led to the discovery of
Rh blood group system is important because of:
1. Hemolytic disease of newborn (HDN) may occur in
Rh-negative pregnant women with Rh-positive fetus.
2. Rh antibodies may develop in Rh-negative patient if
Present Status and Nomenclature
The genes of the Rh system are present on chromosome
1. There are three pairs of genes called Cc, Dd, and Ee
but only five antigens (C, c, D, E, e) as there is no antigen
produced by the ‘d’ gene. Rh gene travels in the set of three,
e.g. CDC, CDE, cDE, etc. with one set being received from
each parent. There are thus, eight possible chromosomes
each of which carries genes for three factors (Table 11.3).
inherited by an individual so that 36 different genotypes
are possible Table 11.4 gives the most common genotypes.
Out of various gene combinations as shown in
Table 11.4, it is presence or absence of D gene which is
most important. When a person inherits ‘D’ antigen its red
cells react with anti-D and these are called Rh-D Positive.
If a person does not inherit D antigen, the red cells do not
react with anti-D and thus, called Rh-D negative.
It is defined as weakened expression of the normal D
antigen, i.e. there are fewer than normal D antigens per red
cells. There are two grades of Du:
High grade Du red cells are agglutinated by certain
anti-D sera while low grade Du are mostly detected by
(Anti-human-globulin) AHG test.
It has been shown that Du positive bloods may immunize
the Rh-negative patient resulting in the formation of anti-D
antibodies, hence, it is important to exclude Du individuals
from Rh negative blood donor list.
Rh antibodies are usually immune IgG type but may be IgM
or even IgA. Most Rh antibodies result from exposure to Rh
positive red cells, either due to pregnancy or transfusion in
individuals who lack the corresponding antigen.
Auto Rh antibodies may be found in individual with
warm autoimmune hemolylic anemia and anti-e is the
Both polyclonal and monoclonal reagents are available in
I. Polyclonal human and D serum
a. Anti-Rh(D) serum for saline or rapid tube test
This contains macromolecular additives and
b. Anti-D for saline tube test
II. Monoclonal anti-D reagents
1. IgM anti-D monoclonal reagent
2. IgG anti-D monoclonal reagent
3. IgM and IgG (Blend) anti-D monoclonal reagent
4. Blend of IgM monoclonal and IgG polyclonal
The IgM anti-D monoclonal is highly specific, saline
reacting working well at room temperature and at 37°C. It
is good for emergency slide test, or immediate spin tube
test as well as routine Rh-D typing, but IgM anti-D are
unreliable for detecting weak D by antiglobulin test, while
IgM and IgG (Blend) monoclonal reagent or IgM anti-D
monoclonal and IgG anti-D (polyclonal) blend can be
used for weak D testing by antiglobulin test.
Rh(D) grouping is done along with ABO grouping using
same techniques as used for ABO grouping:
Slide test is not recommended for routine test because it is
not reliable especially for weak reactive cells.
1. Place one drop of anti-Rh(D) on a labeled slide.
2. Place one drop of 22% albumin on another labeled
3. Put one drop of 40–50% red cells on both the slides.
4. Mix the cell suspension and reagent, using a clean
stick for each slide and spread the mixture evenly on
the slide over area of 15 mm diameter.
5. Place both slides on a view box (lighted), till gently
and continuously for two minutes. Observe for agglutination.
TABLE 11.3: The eight basic chromosomes of fisher and race
Blood Banking (Immunohematology) 333
A positive test has agglutination with anti-Rh(D) in
the ‘test’ and a smooth suspension of cells in control. A
negative test has smooth suspension of cells in both ‘test’
1. Place one drop of anti-Rh(D) serum in a tube labeled
2. Place one drop of 22% albumin in a tube labeled
3. Add 1 drop of 2–5% cell suspension in plasma or serum
4. Mix well and keep at 37°C for 1 hour (sedimentation
In case of emergency, incubate the tube for 10 minutes
at 37°C and then centrifuge at 1000 rpm for 1 minute
5. Gently resuspend the cell button and observe for
agglutination. All negative results must be confirmed
A positive test has agglutination with anti-Rh(D) in the
test and a smooth suspension of cells in the control. A
negative test has smooth suspension of cells in both ‘test’
1. Take one drop of anti-Rh(D) serum in a clean labeled
2. Place one drop of appropriate control in a labelled tube.
3. Add 1 drop of 2–5% cell suspension to be tested to both
4. Mix and incubate both the tubes at 37°C for 15–30
5. Centrifuge at 1000 rpm for 1 minute.
6. Gently resuspend the cell button and examine for
agglutination. If there is strong agglutination of cell in
‘test’ tube, then sample is Rh(D) positive and there is
no need to proceed with antiglobulin phase of test.
7. If no agglutination or doubtful reaction is observed,
wash the cells 3–4 times with saline and decant the
8. Add 1–2 drops of antiglobulin reagent (Coomb’s), mix
gently and centrifuge at 1000 rprn for 1 minute.
9. Resuspend the cell button gently and examine for
agglutination and record the results.
10. If the test is negative, the reaction can be confirmed
by adding known IgG sensitised cells, recentrifuge and
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