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

Eryclone Rh-hr control.

Test Procedure

It is recommended that a negative control be run

simultaneously with each RhoD test sample using

Eryclone Rh-hr control because invalid positive results

may be obtained as with all high-protein blood typing

reagent, especially with samples having autoantibodies or

abnormal serum proteins.

Bring reagent and samples to room temperature before

testing.

Slide Test

 1. Place one drop of Eryclone anti-D (Rho) (lgG) 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 Eryclone anti-D (Rho) (lgG) 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. 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 Eryclone Anti-D (Rho) (lgG) 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.

338 Concise Book of Medical Laboratory Technology: Methods and Interpretations 5. Add two drops of Eryclone anti-human globulin

reagent 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, observing for

agglutination macroscopically.

Interpretation of Results

Slide and Tube Tests

a. Agglutination with reagent and no agglutination with

Rh-hr control is a positive test result and indicates

the presence of the D(Rho) 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 the D(Rho)

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 with a saline reacting

anti-D such as RHOFINAL® anti-D (IgM + IgG).

c. Cord cells heavily sensitized with anti-D(Rho) may

give a false negative immediate spin test result.

Du Test Procedure

a. Agglutination with reagent and no agglutination with

control indicates the presence of Du antigen (weak/

partial Ds). No agglutination with reagent and control

indicates absence of the Du antigen.

b. Mixed field agglutination in the Du test on red cells

from a recently delivered woman may indicate a

mixture of maternal Rho(D) negative and fetal Rho(D)

positive blood,

c. Red cells demonstrating a positive direct antiglobulin

test cannot be accurately tested for Du antigen (weak/

partial Ds).

Remarks

1. As undercentrifugation or overcentrifugation could

lead to erroneous results, it is recommended that

each laboratory calibrates its own equipment and

determine the time required for achieving the desired

results.

2. It is strongly recommended that as a routine quality

control measure known Rho(D) positive and Rho(D)

negative red cells be occasionally run, preferably on a

daily basis so as to control reagent performance and

validation of test results.

3. After usage the reagent should be immediately

recapped and replaced to 2–8°C storage.

ANTI-D (RhO) (IgM + IgG)

Monoclonal Blood Typing Antibodies for

Slide and Tube Tests

(Rhofinal from Tulip)

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 EBV transformation.

Each hybridoma cell line produces homogeneous

antibodies of only one immunoglobulin class, which are

identical in their chemical structure and immunological

activity. Human red blood cells are classified as Rho(D)

positive or Rho(D) negative depending on the presence or

absence of Rho(D) antigen on them. Approximately, 85%

of the Caucasian population is Rho(D) positive. The Du

phenotype is a traditional definition to describe the weak/

partial Ds that can be detected with Rhofinal anti-D

(Rho) (lgM + IgG). About 60% of the Dus(weak/partial Dus)

may react with RHOFINAL anti-D (Rho) (IgM + IgG) in

slide tests and about 90% may be detected by the tube

technique.

Reagent

RHOFINAL anti-D (Rho) (lgM + IgG} is ready-to-use

reagent, prepared from supernatants of cell cultures with

antibody producing B-lymphocytes obtained through EBV

transformation and is a blend of monoclonal antibodies

of immunoglobulin class IgM and IgG. These antibodies

are a mixture of several monoclonal antibodies of the

same specificity but having the capability of recognizing

different epitopes of the human red blood cell antigen D

(Rho).