Interpretation

Agglutination in the ‘test’ tube and none in negative

control tube constitutes a positive test result and blood is

accordingly labeled Rh-Du positive.

ANTI-D (Rho)

Human (IgG) Polyclonal Blood

Typing Antibodies for Slide and

Modified Tube Tests

(Courtesy: Tulip’s Erybank Range)

Summary

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

test.

Reagent

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

antigen D (Rho).

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

modified tube tests.

Reagent Storage and Stability

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.

Principle

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

as described later.

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.

Sample Collection and Storage

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

mentioned time period:

EDTA or heparin : 2 days

Sodium citrate/sodium oxalate : 14 days

ACD or CPD : 28 days

Clotted whole blood should be tested within 14 days.

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, Rl

viewing box with 40–45°C surface temperature, Eryclone

anti-human globulin (Coomb’s) reagent, Eryclone Rh-hr

control.

Test Procedure

Slide Test

1. Place one drop of Erybank anti-D polyclonal reagent

on a clean prewarmed glass slide (40–45°C surface

temperature).

2. Add one equal drop of whole blood.

3. Mix well with a mixing stick uniformly over an area of

approximately 2.5 cm2

.

4. Rock the slide gently, back and forth.

5. Observe for agglutination macroscopically at 2

minutes.

Tube Test

1. Prepare a 5% suspension of the red cells to be tested

in isotonic saline.

2. Place one drop of Erybank Anti-D polyclonal reagent

into a labeled test tube.

3. Pipette into the test tube, one drop of the 5% cell

suspension and mix well.

4. Centrifuge for 1 minute at 1000 rpm (125 g) or 20

seconds at 3400 rpm (1000 g).

5. Gently resuspend the cell button, observing for agglutination macroscopically.

Du Test Procedure

1. Prepare a 5% suspension of the red cells to be tested

in isotonic saline.

2. Place one drop of Erybank anti-D polyclonal reagent

into a labeled test tube.

3. Pipette into the test tube one drop of the 5% cell

suspension and mix well. Incubate at 37°C for 15

minutes.

4. Wash the contents of the tube thoroughly, at least three

times, with isotonic saline and decant completely after

the last wash.

5. Add two drops of Eryclone anti-human globulin

re-agent and mix well.

6. Centrifuge for 1 minute at 1000 rpm (125 g) or 20

seconds at 3400 rpm (1000 g).

7. Very gently, resuspend the cell button and observe for

agglutination macroscopically.

Interpretation of Results

Slide and Tube Tests

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.

Du Test Procedure

a. Agglutination with reagent and no agglutination

with control indicate the presence of Du antigen.

No agglutination with reagent and control indicates

absence of Du antigen.

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

Remarks

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)

Summary

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

class, which are identical in their chemical structure and

immunological activity.