The observations of Levine and Stetson in 1939 and of Landsteiner and

Weiner in 1940 provided the basis for current understanding of the clinical

significance and laboratory detection of Anti-D.

Anti-D monoclonal IgM & IgG reagent has been formulated for use by slide

& tube methods. Use of the reagent by suitable methods will also detect

The transfusion of RhD positive blood to a RhD negative recipient, or

failure to administer a prophylactic to a RhD negative woman can result in

the production of anti-D.

reduction in the expression of their RhD antigen. Other cells may display

qualitative variation in RhD antigen expression, these are referred to as

partial D. Weak D individuals may also be partial D.

The test procedures recommended for the use of this reagent are based

upon the agglutination (clumping) of red blood cells carrying the D antigen

in the presence of an Anti-D antibody.

control (O rr cells) should be tested in parallel with each batch of tests.

The expression of certain red cell antigens may diminish in strength during

storage, particularly in EDTA and clotted samples. Better results may be

obtained with fresh samples.

1. Prolonged incubation during the slide technique may cause drying of

the material, which should not be confused with agglutination.

2. This product should be clear. Turbidity may indicate bacterial

contamination. The reagent should not be used if particles, fibrin gel

or a precipitate are present.

3. The reagents have been characterised by the procedures

recommended in this pack insert and the user must determine their

suitability for use in other techniques. Each reagent has been

optimised for use as supplied by the recommended techniques,

without further dilution or additions.

4. All human components have tested negative for HBsAg, HCV and

HIV 1&2 antibodies. However, as no known test methods offer total

assurance, all material of biological origin should be regarded as a

possible Bio-Hazardous substance and treated with caution by

suitably qualified personnel, in accordance with the guide lines laid

down by the appropriate authorities.

1. SLIDE TECHNIQUE

1.1 Prepare a 30%-45% suspension of test red blood cells in PBS pH 7.0

± 0.2, or autologous plasma or serum.

1.2 Add to a clean, labelled slide:

• One drop of anti-D IgM & IgG blend.

• One drop of the test red cells suspension.

1.3 Mix well by gently and continuously rocking the slide for approx. 30

seconds and incubate the test for 5 minutes at room temperature,

with occasional mixing.

1.4 Examine macroscopically for agglutination. A diffuse light source may

aid reading.

2. TUBE TECHNIQUE – IMMEDIATE SPIN

2.1 Prepare a suspension of test red cells 2-3% in PBS pH7.0 ± 0.2 or

1.5-2% in LISS.

2.2 Place in a small, labelled test tube:

• 1 volume of anti-D IgM & IgG blend.

• 1 volume of suspended red cells.

2.3 Mix well.

2.4 Centrifuge immediately for 10 seconds at 1000g or for a suitable

alternative force and time.

2.5 Agitate the tube gently to dislodge the cell button and examine

macroscopically for agglutination.

3. TUBE TECHNIQUE – LISS

3.1 Place in a small, labelled test tube:

• 1 volume of Biotec blood grouping reagent

• 1 volume of red cells suspended 1.5-2% in LISS.

3.2 Mix well and incubate for 15-20 minutes at 37°C.

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3.3 Centrifuge immediately for 10 seconds at 1000g or for a suitable

alternative force and time.

3.4 Agitate the tube gently to dislodge the cell button and examine

macroscopically for agglutination.

After reading the immediate spin results, re-incubate the test for a

described below.

OR

further incubation, following the procedure given below.

4.1 Wash the test 4 times with a large excess of PBS pH7.0 ± 0.2 (e.g.

4ml of PBS per 12 (or 10) x 75mm glass tubes).

b) make sure that most of the residual saline is removed at the

end if each wash to leave a ‘dry’ cell button.

4.2 Add two drops of anti-human globulin reagent to each tube.

4.3 Mix thoroughly.

4.4 Centrifuge at 1000g for 10 seconds or for a suitable alternative force

and time.

4.5 Agitate the tube gently to dislodge the cell button and examine

macroscopically for agglutination.

This reagent contains 1g/L sodium azide, which may be toxic if ingested. It

may also react with lead and copper plumbing to form explosive salts. On

disposal, flush with large quantities of water. The formulation also contains

EDTA.

1. Store at 2-8ºC.

2. DO NOT FREEZE.

3. Allow reagents and samples to reach room temperature prior to use.

4. The expiry date is printed on the bottle label.

1. Slide tests are not recommended for the detection of weak or partial

D. All slide tests should be confirmed by tube grouping.

2. Tube tests should be read by a ‘tip and roll’ procedure. Excessive

agitation may disrupt weak agglutination and produce false negative

results.

3. Driblocks and waterbaths promote better heat transfer and are

recommended for 37°C testing, particularly where the incubation

period is 30 minutes or less.

4. Some weak and/or partial D samples will not react with this reagent.

5. The expression of certain red cell antigens may diminish in strength

during storage, particularly in EDTA and clotted samples. Better

results will be obtained with fresh samples.

6. Certain tests performed on unwashed samples (e.g. cord) or samples

stored and tested at 20°C or below, may exhibit false positive

reactions due to the formulation of this reagent.