RHOFINAl

 Anti-D (Rho) (IgM + IgG) is a blend of IgM

and IgG class of Anti-D (Rho) monoclonals, a characteristic

which accords versatility to the reagent. It gives an avid

saline reacting slide/tube test reagent the capability of

detecting Du (weak/partial Ds) in the anti-human globulin

phase. Each batch of reagent undergoes rigorous quality

control at various stages of manufacture for its specificity,

avidity and performance.

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.

Blood Banking (Immunohematology) 339

Principle

Human red blood cells possessing D(Rho) antigen

will agglutinate in the presence of antibody directed

towards the antigen. Agglutination of red blood cells with

RHOFINAL anti-D (Rho) (IgM-IgG) reagent is a positive

test result and indicates the presence of D(Rho) antigen.

No agglutination with RHOFINAL anti-D (Rho) (IgM

+ IgG) reagent is a negative test result and indicates the

absence of D(Rho) antigen. All negative test results should

be further tested for Du (weak/partial Ds) by performing

the Du test procedure, as described later.

Note

1. In vitro diagnostic reagent for laboratory and

professional use only. Not for medicinal use.

2. RHOFINAL anti-D (Rho) (lgM + IgG) 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 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

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 (12 × 75 mm), Pasteur

pipettes, isotonic saline, centrifuge, timer, mixing sticks,

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

Test Procedure

Bring reagent and samples to room temperature before

testing.

Slide Test

1. Place one drop of RHOFINAL anti-D (Rho) (lgM +

lgG) 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 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 RHOFINAL anti-D (Rho) (IgM +

IgG) reagent into labeled test tube.

3. Pipette into each of 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 RHOFINAL anti-D (Rho) (lgM +

lgG) reagent into labeled test tube.

3. Add to the test tube, one drop of the cell suspension

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

4. Wash the contents of the tube, 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 the D(Rho) antigen. Do not interpret

peripheral drying or fibrin strands as agglutination.

No agglutination is a negative test result and indicates

absence of the D(Rho) antigen.

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

give a false negative immediate spin test result.

Du Test Procedure

a. Agglutination with the reagent and no agglutination

with the control indicate presence of the Du antigen

(weak/partial Ds). No agglutination with reagent and

control indicates absence of the Du antigen (weak/

partial Ds).

340 Concise Book of Medical Laboratory Technology: Methods and Interpretations 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).