RH Debil Du

Vol 4 (2), July 2013; 16-19
Detection of Du Antigen in Rh Negative Blood Group Individuals
Naheed Afshan1*, Sarah Tariq1
1Department of Microbiology, Jinnah University for Women, Karachi -74600, Pakistan
ABSTRACT
Du is the weak expression of D antigen. The cells which are not immediately agglutinated by Anti-D
sera cannot be easily classified as D negative because some of these agglutinate after addition of
antiglobulin sera. This weak reactivity is termed as Du. Du positive cells are likely to elicit an immune
response in D negative individuals and the Du cells could be destroyed if the recepient is already
immunized. Therefore, Du positive donor is treated as D positive and recipient is treated as negative.
This report is based on Du antigen and the testing of Du antigen. In this report we discussed about
the detection of Du Antigen using 2 different protocols that show how to test the presence and absence
of Du antigen in Rh negative blood group individuals. In this study we included 100 blood group D-
negative individuals .The result showed there were 3% that have Du antigen in their blood.
Keywords: Agglutination, Blood group, Du antigen, Rh antigen, Serum
INTRODUCTION negative person is found to have anti-D, that

individual has invariably been exposed to Rh positive
The Rh system includes many antigens but the major cells. The two most likely ways for Rh positive red
one is D, alternatively reffered as Rho. The term Rh cells to reach the circulation of an Rh negative
positive is used to denote red cells that carry the D individual are: (1) Transfusion of red cells from an
(Rh) antigen or its variant Du. Red cells that have Rh positive donor to an Rh negative recipient. Except
neither D nor Du on their membranes are termed Rh in rare circumstances, this is contrary to good
negative. With the exception of A and B, the most transfusion practice; therefore it is usually the result
important of all blood group antigens is undoubtedly of clerical or technical error, (2) Passage of red cells
D. This is because the consequence of the presence from an Rh positive foetus through the placenta to
can be severe and Rh haemolytic disease of the the Rh negative mother. This almost always occurs
newborn can be tragic: transfusion reaction due to to some extent at delivery and occasionally late in
Rh antibodies can be heartbreaking experience. pregnancy.
However, unlike the situation in the ABO system,
an Rh negative person does not usually have anti- Du Antigen: In transfusion medicine, after the ABO
D in his or her serum. Rh antigens are confined to blood groups, the D antigen is the most significant.
red cells and are not found in body fluids or natural A high pro¬portion of people whose red blood cells
substances; therefore, exposure to red cells is the (RBCs) lack D will make anti-D if exposed to the
only way a person can become immunised to Rh. D antigen by pregnancy or transfusion. Accordingly,
Also contributing to the importance of the Rh system all D– patients, especially girls and women who may
is the fact that the D antigen is one of the most become pregnant, should be transfused with D–
effective blood group immunogens. As stated above, RBCs. The D antigen is in the Rh blood group
no natural substances chemically similar to the sys¬tem, which with 49 distinct antigens is the most
D antigen have been found; therefore when an Rh polymorphic blood group system. This document
*Corresponding author: [email protected] reviews fundamental in¬formation for the D antigen.
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Vol 4 (2), July 2013; 16-19
testing the red cells of recipients for Du is considered

Du is the phenotypic term used to denote a weakened unnecessary. The recipient's welfare is not
expression of the D antigen. Du originally defined compromised if he or she is of the Du phenotype
as those red cells reacting with anti-D only when a but is typed as D negative and receives Rh negative
more sensitive indirect antiglobulin test was used. red cells. In such circumstances Rh negative donor
Du phenotype can arise from three different genetic blood may be used unnecessarily.
situations. a) A person may inherit a gene coding for
weakened quantitative expression of D antigen. b) It is important that the Du status of the D negative
One gene may interact with another to modify and pregnant woman be established early in pregnancy.
weaken the expression of the D antigen. c) A gene If the mother is found to be Rh positive, Du varient,
may not code for the total material that makes up she is not a canditate for Rh immunoglobulin
the antigen.The frequency of Du antigen is relatively prophylaxis- either antepartum or postpartum-
low less than 1%. Du is a poor immunogen, however, whereas the Rh negative (D and Du negative) mother
accelerated destruction of Du red cells can result if is a canditate. The reason for performing the Du test
transfused to a person already making anti-D. Hence early in pregnancy is to avoid mis-interpreting the
Du donor units are currently labelled as Rah positive. cause of a positive fetal cell screening test at the
Du recipients are labelled as Rah negative. Newborn time of delivery.
of Rah negative mother are tested for D & Du and
Rh Ig is recommended for mothers of D positive or In addition to prenatal patients, newborn babies are
Du positive infants in order to prevent potential also tested for Du if they type as D negative. Again,
immunisation. The terms Du variant or partial Du this relates to the need for Rh immune globulin: the
are recommended when there is both a qualitative D negative, Du negative baby cannot immunize its
and quantitative difference noted in the D antigen. mother; for this reason she does not need Rh immune
globulin protection. However, the mother should
Du Testing: Not all red cells can be classified as Rh receive Rh immune globulin if the baby is of the Du
positive or negative by direct agglutination tests. phenotype.
The cells of a few persons react weakly with anti-
D or requires a longer reaction time than most Rh Du red cells fall into a wide spectrum of reactivity
positive cells. An even smaller number of persons when tested with anti-D reagents. How each cell is
have red cells that are not agglutinated by Not Not detected depends on the type of anti-D that is used
all red cells can be classified as Rh positive or and the kind of test that is performed. To test for Du,
negative by direct agglutination tests. The cells of red cells are incubated at 37oC with an IgG anti-D
a few persons react weakly with anti-D or requires and an antiglobulin test is performed. If serum
a longer reaction time than most Rh positive cells. suspended cells are used, some blood samples at the
antiglobulin serum. An even smaller number of upper end of the Du spectrum will be agglutinated
persons have red cells that are not agglutinated by weakly by most anti-D reagents prior to the
antiglobulin serum. These cells are called Du. Cells antiglobulin test, either at room temperature or at
of the Du phenotype may fall anywhere within this 37oC. When the same red cells are suspended in
spectrum of reactivity with anti-D. saline, direct agglutination may not be observed, or
it may be seen with one reagent and not another.
Because Du is a form of D, red cells of the Du Regardless of whether the red cells are agglutinated
phenotype can stimulate the production of anti-D in directly by anti-D or they absorb anti-D and it is
Rh negative recipients and, more importantly, react detected in the antiglobulin phase of the test, they
with anti-D in vivo. It is for these reasons that donor are Rh positive, provided both controls for D typing
blood must be shown to be negative not only in the and the Du test are negative.
test for D but also in the test for Du. In general,
17
Vol 4 (2), July 2013; 16-19
MATERIALS & METHODS removed from the cell suspension. Add 1-2 drops of
coombs reagent in the tube containing washed red
Blood Sample: Any blood group sample with EDTA cells. After addition of coombs reagent, centrifuge
which is Rh-negative. the tube at 3500 rpm for 15 seconds. Immediately
resuspend the tube and examine for agglutination
Reagents: Anti-D antisera, 3-5% Red cell suspension, using Electron microscope. Confirm all negative
Normal saline, Coombs reagent, 37 C incubator, results by adding one drop coombs control cells to
Albumin. all tubes showing no agglutination and centrifuge15-
30 seconds at 3500 rpm. Gently resuspend & examine
Procedures: for agglutination. Agglutination should be present
in this step or the test is invalid.
i) Without Albumin: Prepare a washed, 3-5%
suspension of RBCs. Add 50ul Anti D in a tube OBSERVATION
containing 50ul 3-5% Red cell suspension. Incubate
the tube at 37 C for 40-45 minutes. After 40-45 min, Table I. How to read the result.
suspend the tube & examine agglutination, if
agglutination occurs it means Rh is positive & if no
agglutination present it confirmed that Rh is negative.
Centrifuge the suspension at 3500 rpm for 15 seconds.
After centrifugation, washed the suspension 3 times
with normal saline. After 3rd time washing, tapping
should be done so that all the remaining saline should
be removed from the cell suspension. Add 1-2 drops
of coombs reagent in the tube containing washed
red cells. After addition of coombs reagent, centrifuge
the tube at 3500 rpm for 15 seconds. Immediately
resuspend the tube and examine for agglutination
using Electron microscope. Confirm all negative
results by adding one drop coombs control cells to
all tubes showing no agglutination and centrifuge15-
30 seconds at 3500 rpm. Gently resuspend & examine
for agglutination. Agglutination should be present
in this step or the test is invalid.
ii) With Albumin: Prepare a washed, 3-5%

suspension of RBCs. Add 50ul Anti D & 1-2 drops Table II. Results showing presence and absence of Du
Albumin in a tube containing 50ul 3-5% Red cell antigen.
suspension. Incubate the tube at 370C for 40-45
minutes. After 40-45 min, suspend the tube &
examine agglutination, if agglutination occurs it
means Rh is positive & if no agglutination present
it confirmed that Rh is negative. Centrifuge the
suspension at 3500 rpm for 15 seconds. After
centrifugation, washed the suspension 3 times with
normal saline. After 3rd time washing, tapping should
be done so that all the remaining saline should be
18
Vol 4 (2), July 2013; 16-19
RESULTS & DISCUSSION CONCLUSION
100 blood samples taken from different individuals From the result, it is concluded that there were rare
having different blood groups but all were Rh cases in peoples that have Du antigen in their blood.
negative individuals. From 100 samples, 3 were Du We cannot avoid to detect Du antigen before
positive. transfusion because if Donor is Rh negative and
Recipient is also Rh negative but Du antigen is
As described earlier, people whose RBCs have a present in the donor blood so if we can’t test Du
weak D phenotype (quantitative D variant) do not antigen mismatched in transfusion occur which will
make anti-D, whereas people whose RBCs have a results in mild to life threatening complications or
partial D phenotype (qualitative D variant with or death.
without weakening of the D antigen) can make
alloanti-D. This presents a different problem REFERENCES
depending on whether the person is a donor or a
patient. For donors, detection of weak and partial D Reid ME and Lomas-Francis C. 2004. Blood Group
antigens would eliminate the possibility of Antigen FactsBook. 2nd ed. San Diego: Academic
immunization should such blood be transfused to a Press.
true D-negative patient. However, historical data
show that weakly expressed D antigens are most Reid ME and Mohandas N. 2004. Red blood cell
unlikely to be immunogenic. blood group antigens: Structure and function. Semin
Hematol, 41: 93-117.
Clinical complications result from RBC destruction
due to the interaction of an alloantibody with RBCs Reid ME and Yazdanbakhsh K. 1998. Molecular
carrying the corresponding antigen. The D antigen insights into blood groups and impli¬cations for
is highly immunogenic and induces an immune blood transfusions. Curr Opin Hematol, 5:93-102.
response in 80% of D-negative persons when
transfused with 200 mL of D-positive blood. For Tayyab M, Malik AR and Khan AS. 2000. Du
this reason, in most countries D typing is performed phenotype - a review. JAMC,12(3): 41-44.
routinely on every blood donor and transfusion
recipient so that D-negative patients receive D- Westhoff CM. 2007. The structure and function of
negative RBC products. Consequently, clinical the Rh antigen complex. Semin Hematol, 44:42-50.
complications due to mismatched transfusions are
infrequent. In contrast, despite the use of
immunosuppressive therapy with anti-D
immunoglobulin prophylaxis, D alloimmunization
in pregnancy still occurs.
Patients with acute or chronic myeloid leukemia,

myeloid metaplasia, polycythemia, or myelofibrosis
occasionally have 2 populations of RBCs of different
Rh type. In some cases, a loss of Rh antigens is
associated with chromosome aberrations.
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Vol 4 (2), July 2013; 16-19

Detection of Du Antigen in Rh Negative Blood Group Individuals

Naheed Afshan1*, Sarah Tariq1

1Department of Microbiology, Jinnah University for Women, Karachi -74600, Pakistan

Keywords: Agglutination, Blood group, Du antigen, Rh antigen, Serum

INTRODUCTION negative person is found to have anti-D, that

testing the red cells of recipients for Du is considered

ii) With Albumin: Prepare a washed, 3-5%

RESULTS & DISCUSSION CONCLUSION

Patients with acute or chronic myeloid leukemia,

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