Human red blood cells are classified as RhoD positive or

RhoD negative depending on the presence or absence of

D antigen on them. Approximately, 85% of the Caucasian

population is Rh positive. The Du phenotype is a variant of

D antigen and is recognized by performing the antiglobulin

test.

About 60% of the traditional Dus, now classified as

weak or partial D’s may react with Eryclone anti-D (IgM)

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

technique.

Reagent

Eryclone anti-D (IgM) is a 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. These antibodies are a

mixture of several monoclonal antibodies of the same

specificity but having the capability of recognizing

different epitopes of human red blood cell antigen D

(Rho).

Eryclone Anti-D (IgM) does not detect all weak and

partial D’s. For the confirmation of negative reactions with

Eryclone Anti-D (IgM) further testing with an incomplete

Anti-D of IgG class is strongly recommended to confirm

the presence or absence of weak/partial D’s.

Each batch of reagent undergoes rigorous quality

control at various stages of manufacture for its specificity,

avidity and performance.

Reagent Storage and Stability

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.

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 Eryclone

anti-D (IgM) reagent is a positive test result and indicates

the presence of D antigen. No agglutination with the

reagent is a negative test result and indicates the absence

of D antigen. All negative test results should be further

tested for D (presence of weak/partial Ds) by performing

the Du test procedure using an incomplete Anti-D of IgG

class, as described later.

Note

1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.

2. Eryclone Anti-D (IgM) reagent is not from human

source, hence, contamination due to HBsAg and HIV

is practically excluded.

3. The reagent contains sodium azide 0.1% as preservative.

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 or 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, eryclone anti-human globulin (Coomb’s) reagent,

Eryclone anti-D (lgG) or RHOFINAL anti-D (lgM + lgG).

336 Concise Book of Medical Laboratory Technology: Methods and Interpretations Test Procedure

Bring reagent and samples to room temperature before

testing.

Slide Test

1. Place one drop of Eryclone anti-D (lgM) reagent on a

clean glass slide

2. Pipette one equal drop of whole blood on the slide.

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

approximately 2.5 cm.

4. Rock the slide gently, back and forth.

5. Observe for agglutination macroscopically at two

minutes.

Immediate Spin 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 (lgM) 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 any incomplete anti-D (IgG class)

reagent such as Eryclone anti-D (IgG) into a labeled

test tube.

3. Add to 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

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 and observe for

agglutination macroscopically.

Interpretation of Results

Slide and Tube tests

a. Agglutination is a positive test result and indicates the

presence of D antigen. Do not interpret peripheral

drying or fibrin strands as agglutination. No agglutination is a negative test result and indicates the

absence of D antigen.

b. Cord cells heavily sensitized with anti-D may give a

false negative immediate spin test result.

Du Test Procedure

a. Agglutination indicates the presence of Du antigen

(Presence of weak/partial Ds). No agglutination indicates

the absence of Du antigen (Absence of weak/partial Ds).

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

from a recently delivered woman may indicate a

mixture of maternal RhoD negative and fetal RhoD

positive blood.

c. Red cells demonstrating a positive direct antiglobulin

test cannot be accurately tested for Du antigen

(Presence of weak/partial Ds).

Remarks

As under centrifugation or overcentrifugation could

lead to erroneous results, it is recommended that each

laboratory calibrate its own equipment and determine

the time required for achieving the desired results. It is

strongly recommended that as a routine quality control

measures known RhoD positive and RhoD negative red

cells be occasionally run, preferably on a daily basis so as

to control reagent performance and validate test results.