Agglutination in the ‘test’ tube and none in negative
control tube constitutes a positive test result and blood is
accordingly labeled Rh-Du positive.
Typing Antibodies for Slide and
(Courtesy: Tulip’s Erybank Range)
Polyclonal antibodies are derived from hyperimmune
human serum containing Anti-D (Rho) antibodies directed
towards the human D (Rho) antigen. Human red blood
cells are classified as RhoD positive or RhoD negative
depending on the presence or absence of the D antigen
on them. Approximately 85% of the Caucasian population
is RhoD positive. The Du phenotype is a variant of the D
antigen and is recognized by performing the antiglobulin
Erybank anti-D polyclonal is a ready-to-use high-protein
reagent prepared from pools of hyperimmune human
serum containing antibodies directed towards the human
D (Rho) antigen. These antibodies of the immunoglobulins
class IgG are a mixture of several polyclonal antibodies
of the same specificity but having the capability of
recognizing different epitopes of the human red blood cell
Each batch of reagent undergoes rigorous quality
control at various stages of manufacture for its specificity,
avidity and titer. The reagent is suitable for slide and
1. Store the reagent at 2–8°C. Do not freeze.
2. The shelf life of the reagent is as per the expiry date
mentioned on the reagent vial label.
Human red blood cells possessing D antigen will
agglutinate in the presence of antibody directed towards
the antigen. Agglutination of red blood cells with Erybank
anti-D polyclonal reagent is a positive test result and
indicates the presence of D antigen. No agglutination
with anti-D reagent is a negative test result and indicates
absence of D antigen. All negative test results should be
further tested for Du by performing the Du test procedure
334 Concise Book of Medical Laboratory Technology: Methods and Interpretations Note
1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.
2. Erybank anti-D polyclonal reagent is from human
source and the source material used in its manufacture
is tested by approved techniques and found negative
for HBsAg and HIV, HCV antibodies.
3. The reagent contains sodium azide 0.1% as preser -
vative. Avoid contact with skin and mucosa. On
disposal flush with large quantities of water.
4. Extreme turbidity may indicate microbial contamination or denaturation of protein due to thermal
damage. Such reagent should be discarded.
No special preparation of the patient is required prior
to sample collection by approved techniques. Samples
should be stored at 2–8°C if not tested immediately. Do
not use hemolyzed samples. Anticoagulated blood using
various anticoagulants should be tested within the below
Sodium citrate/sodium oxalate : 14 days
Clotted whole blood should be tested within 14 days.
Additional Material Required for Slide and Tube
Glass slides (50 × 75 mm), test tubes (10 × 75 mm), Pasteur
pipettes, isotonic saline, centrifuge, timer, mixing sticks, Rl
viewing box with 40–45°C surface temperature, Eryclone
anti-human globulin (Coomb’s) reagent, Eryclone Rh-hr
1. Place one drop of Erybank anti-D polyclonal reagent
on a clean prewarmed glass slide (40–45°C surface
2. Add one equal drop of whole blood.
3. Mix well with a mixing stick uniformly over an area of
4. Rock the slide gently, back and forth.
5. Observe for agglutination macroscopically at 2
1. Prepare a 5% suspension of the red cells to be tested
2. Place one drop of Erybank Anti-D polyclonal reagent
3. Pipette into the test tube, one drop of the 5% cell
4. Centrifuge for 1 minute at 1000 rpm (125 g) or 20
5. Gently resuspend the cell button, observing for agglutination macroscopically.
1. Prepare a 5% suspension of the red cells to be tested
2. Place one drop of Erybank anti-D polyclonal reagent
3. Pipette into the test tube one drop of the 5% cell
suspension and mix well. Incubate at 37°C for 15
4. Wash the contents of the tube thoroughly, at least three
times, with isotonic saline and decant completely after
5. Add two drops of Eryclone anti-human globulin
6. Centrifuge for 1 minute at 1000 rpm (125 g) or 20
7. Very gently, resuspend the cell button and observe for
agglutination macroscopically.
a. Agglutination with reagent and no agglutination
with control is a positive test result and indicates
the presence of D antigen. Do not interpret peripheral drying or fibrin strands as agglutination. No
agglutination with reagent and control is a negative
test result and indicates absence of D antigen.
b. Agglutination in Rh-hr (negative) control indicates the
presence of autoantibodies or rouleaux formation. In
such cases, it is recommended that the determination
of Rh factor should be made in a saline reacting anti-D
such as RHOFINAL anti-D (IgM + IgG).
c. Cord cells, heavily sensitized with anti-D, may give a
false negative immediate spin test result.
a. Agglutination with reagent and no agglutination
with control indicate the presence of Du antigen.
No agglutination with reagent and control indicates
b. Mixed field agglutination in Du test on red cells from
a recently delivered woman may indicate a mixture of
maternal Rh negative and fetal Rh positive blood.
c. Red cells demonstrating a positive direct antiglobulin
test cannot be accurately tested for Du antigen.
Blood Banking (Immunohematology) 335
As undercentrifugation or overcentrifugation could lead to
erroneous results, it is recommended that, each laboratory
calibrates its own equipment and the time required for
achieving the desired results.
ANTI-D (RhO) (IgM) Monoclonal Blood
Typing Antibodies for Slide and Tube Tests
(Courtesy: Tulip’s Eryclone Range)
Monoclonal antibodies are derived from hybridoma cell
lines, created by fusing mouse antibody producing B
lymphocytes with mouse myeloma cells or are derived
from a human B cell line through Epstein–Barr Virus
(EBV) transformation. Each hybridoma cell line produces
homogeneous antibodies of only one immunoglobulin
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