Sample Collection and Preparation
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 or 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 (12 × 75 mm), pasteur
pipettes, isotonic saline, centrifuge, timer, mixing sticks.
Bring all reagent and samples to room temperature before
1. Prepare a 10% suspension of the red blood cells to be
2. Place one drop of Erybank anti-A1 lectin on a clean
3. Pipette two drops of the cell suspension on the slide.
4. Mix well with a mixing stick uniformly over an area of
5. Rock the slide gently, back and forth.
6. Observe for agglutination macroscopically at one
1. Prepare a 5% suspension of the red cells to be tested
2. Place one drop of Erybank anti-A1 lectin into a labeled
3. Pipette into the test tube, one drop of the test red cell
4. Centrifuge for 1 minute at 1000 rpm (125 g) or 20
5. Gently resuspend the cell button, observing for
agglutination macroscopically.
Agglutination is a positive test result and indicates the
presence of A1 antigen. Do not interpret peripheral drying
or fibrin strands as agglutination. No agglutination is a
negative test result and indicates the absence of A1 antigen.
1. A1 antigen is not fully expressed on the red blood
cells of newborns below one year of age, hence, false
2. It is strongly recommended that known A1 and A2
cells should be occasionally run, preferably on a daily
basis to control reagent performance and validate test
3. A1 A2 (Aint) cells may agglutinate moderately with
Erybank®. Anti-A1 lectin. These should be tested further
with Anti-H lectin to confirm Aint cells.
4. As undercentrifugation or overcentrifugation can lead
to erroneous results, it is recommended that each
laboratory calibrate its own equipment and determine
the time required for achieving the desired result.
(Courtesy: Tulip’s Erybank Range)
The H antigen is a basic blood group antigen present in
human beings. There is considerable variation in the H
antigen content in different individuals of the same ABO
group, but the general pattern indicates their strength as
O>A2>A2B>B>A1>A1B. Water-soluble H substance can also
be demonstrated in saliva or body fluids of individuals
agglutinate with Anti-H lectin are classified as Bombay
phenotype (Oh). The Bombay phenotype is more common
in India than other parts of the world and the estimated
gene frequency of Oh phenotype in Bombay is 0.0066%.
Erybank Anti-H lectin is a ready-to-use purified extract of
Ulex europaeus seeds. It contains a phytohemagglutinin
which is virtually specific for the H antigen on human red
blood cells. Erybank Anti-H lectin is used for recognition
of the H antigen on human red blood cells. It is useful,
especially for assessing the H secretor status of group
‘O’ individuals and also in differential grouping of Aint
subgroup along with anti-A lectin.
a. Store the reagent at 2–8°C. Do not freeze.
b. The shelf life of the reagent is as per the expiry date
mentioned on the reagent vial label.
Human red blood cells possessing the H antigen will
agglutinate in the presence of seed extract (lectins)
containing phytohemagglutinin specifically directed
towards it. Water-soluble H substance present in saliva
neutralizes anti-H lectin. Agglutination of red blood cells/
neutralization of anti-H lectin by saliva is a positive test
result and indicates the presence of H substance on/in the
red cell/saliva respectively. No agglutination/neutralization
of anti-H lectin is a negative test result and indicates the
absence of H substance on/in the red cell/saliva respectively.
1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.
2. The reagent contains sodium azide 0.1% as preservative.
Avoid contact with skin and mucosa. On disposal flush
with large quantities of water.
3. Extreme turbidity may indicate microbial contamination/reagent deterioration. Such reagent should
Sample Collection and Preparation
For Recognition of H Antigen on Human Red Blood Cells
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 or sodium oxalate : 14 days
Clotted whole blood should be tested within 14 days.
For Assessing Secretor Status in Human Saliva
a. Collect about 2 mL of fresh saliva in a glass tube and
incubate in a boiling water bath for 10 minutes.
b. Centrifuge at 3400 rpm (1000 g) for 10 minutes.
c. Use the clear supernatant immediately for the study
or freeze immediately if to be tested later.
Additional Material Required for Slide and Tube Tests
Glass slides (50 × 75 mm), test tubes (10 × 75 mm), Pasteur
pipettes, isotonic saline, centrifuge, timer, mixing sticks,
red blood cells positive for H antigen, red blood cells
negative for H antigen, saliva positive for H antigen, Saliva
Bring all reagent and samples to room temperature before
1. Place one drop of Erybank anti-H lectin on a clean
2. Add one drop of whole blood to be tested on the slide
and mix well with a mixing stick uniformly over an area
3. Rock the slide gently, back and forth.
4. Observe for agglutination macroscopically at 2 minutes.
1. Prepare a 5% suspension of the red cells to be tested
2. Place one drop of Erybank anti-H lectin into a test tube.
3. Pipette into the test tube, one drop of the test red cell
4. Centrifuge for 1 minute at 1000 rpm (125 g) or 20
5. Gently resuspend the cell button, observing for
agglutination macroscopically.
1. Place two drops of anti-H lectin into two clean glass
2. Pipette two drops of saliva into the tubes and mix well.
3. Incubate at room temperature for 10 minutes.
4. Add one drop of negative and positive cell suspensions
into the tubes, mix well and incubate at room temperature for 5 minutes.
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