Store 2 - 8ºC.
PRINCIPLE OF THE METHOD
When used by the recommended techniques, the reagent will not affect the first stage of
haemagglutination (antibody uptake) but it will enhance the second stage (agglutination) by
allowing the antibody-coated red cells to come closer together than they would in a saline
medium without additives (see Limitations).
REAGENTS
Spinreact 22% and 30% Serological Albumin are prepared from a mixture of bovine serum
albumin, and buffered saline. The polymer content of the Polymer Enhanced BSA is increased
naturally by a process modification. No artificial avidity enhancers or high molecular weight
agglutination potentiators are added to any BSA preparation. None of the BSA reagents do
contain sodium caprylate. Each BSA reagent is supplied at optimal dilution for use with all the
recommended techniques stated below without the need for further dilution or addition. For lot
reference number and expiry date see Vial Labels.
PRECAUTIONS
1. The reagents are intended for in vitro diagnostic use only.
2. If a reagent vial is cracked or leaking, discard the contents immediately.
3. Do not use the reagents past the expiration date (see Vial Label).
4. Do not use the reagents if a precipitate is present.
5. Protective clothing should be worn when handling the reagents, such as disposable
gloves and a laboratory coat.
6. The reagents have been filtered through a 0.2 µm capsule to reduce the bio-burden.
Once a vial has been opened the contents should remain viable up until the expiry date as
long as there is no marked turbidity, which can indicate reagent deterioration or
contamination.
7. The BSA has been obtained from a closed herd in the female line since 1980, in
which no animal has been clinically suspected of having Bovine Spongiform
Encephalopathy (BSE), and which has not been fed rations containing ruminant derived
protein during that period.
8. The reagents contain 0.1% sodium azide. Sodium azide may be toxic if ingested and
may react with lead and copper plumbing to form explosive metal azides. On disposal
flush away with large volumes of water.
9. For information on disposal of the reagents and decontamination of a spillage site
see Material Safety Data Sheets, available on request.
NOTES
1. Red cells sensitised with an in vitro or in vivo autoantibody may agglutinate spontaneously
in concentrations of serological albumin as low as 6%. It is therefore essential to routinely set up
control tests in which the test red cells are mixed with the appropriate serological albumin solution
alone.
2. The antiglobulin techniques can only be considered valid if all negative tests react positively
with IgG sensitised red cells.
3. In the Recommended Techniques one volume is approximately 40µl when using the vial
dropper provided.
4. The use of the reagents and the interpretation of results must be carried out by properly
trained and qualified personnel in accordance with the requirements of the country where the
reagents are in use.
5. The user must determine the suitability of the reagents for use in other techniques.
STORAGE
Do not freeze. Reagent vials should be stored at 2 - 8ºC on receipt. Prolonged storage at
temperatures outside this range may result in accelerated loss of reagent reactivity.
Reagent will remain stable for up to 7 days when subjected to temperatures not
exceeding 30ºC.
MATERIAL REQUIRED
Anti-human globulin or anti-IgG.
Coombs cell washer.
Glass test tubes (10 x 75 mm or 12 x 75 mm).
IgG sensitised red cells.
Spinreact Inert AB serum.
Phosphate Buffered Saline (PBS): NaCl 0.9%, pH 7.0 ± 0.2 at 22ºC ± 1ºC
Test tube centrifuge.
Volumetric pipettes.
Water bath or dry heat incubator equilibrated to 37ºC ± 2ºC.
SAMPLES
Blood samples should be drawn aseptically into EDTA and tested within 48 hours. If
EDTA is unavailable, samples drawn into ACD, CPD or CPDA-1 are acceptable and may
be tested up to 35 days form the date of withdrawal. All blood samples should be washed
at least twice with PBS before being tested.
PROCEDURE
A. Albumin Immediate Spin Technique
1. Prepare a 2-3% suspension of washed test red cells in PBS.
2. Place in a labelled test tube: 2 volumes each of test serum, test red cell suspension
and 22% Serological Albumin.
3. Mix thoroughly and centrifuge all tubes for 20 seconds at 1000 rcf or for a suitable
alternative time and force.
4. Examine supernatant for haemolysis, then gently resuspend cell button and examine
macroscopically for agglutination.
B. Albumin Room Temperature Saline Phase Technique
1. Prepare a 2-3% suspension of washed test red cells in PBS.
2. Place in a labelled test tube: 2 volumes test serum, 1 volume test cell suspension and 2 volumes 22% Serological Albumin.
3. Mix thoroughly and incubate at 18-25ºC for 5-30 minutes.
4. Centrifuge all tubes for 20 seconds at 1000 rcf or for a suitable alternative time
and force.
5. Examine supernatant for haemolysis, then gently resuspend cell button and
examine macroscopically for agglutination.
C. Albumin 37ºC Technique
1. Prepare a 2-3% suspension of washed test red cells in PBS.
2. Place in a labelled test tube: 2 volumes test serum, 1 volume test cell suspension
and 2 volumes 22% Serological Albumin.
3. Mix thoroughly and incubate at 37ºC for 15-60 minutes.
4. Centrifuge all tubes for 20 seconds at 1000 rcf or for a suitable alternative time
and force.
5. Examine supernatant for haemolysis, then gently resuspend cell button and
examine macroscopically for agglutination.
D. Indirect Antiglobulin Technique (IAT)
1. Follow steps 1 to 3 of Albumin 37ºC Technique above.
2. Wash test red cells 4 times with PBS, taking care to decant saline between
washes and resuspend each cell button after each wash. Completely decant saline
after last wash.
3. Add 2 volumes of anti-human globulin to each dry cell button.
4. Mix thoroughly and centrifuge all tubes for 20 seconds at 1000 rcf or for a suitable
alternative time and force.
5. Gently resuspend red cell button and read macroscopically for agglutination.
E. Antibody Titration Technique
1. Prepare a 2-3% suspension of washed test red cells in Spinreact 22% Serological
Albumin.
2. Prepare doubling dilutions of test serum in inert AB serum.
3. Add 1 volume of test red cell suspension to 1 volume of each dilution.
4. Mix thoroughly and incubate at 37ºC for 15-60 minutes.
5. Centrifuge all tubes for 20 seconds at 1000 rcf or for a suitable alternative time
and force.
6. Gently resuspend each cell button and read macroscopically for agglutination.
INTERPRETATION OF TEST RESULTS
1. Positive: Agglutination of test red cells constitutes positive test result within
accepted limitations of the test procedure.
2. Negative: No agglutination of the test red cells constitutes negative test result
within accepted limitations.
Stability of the reactions
1. Tube tests should be read immediately after centrifugation.
2. Washing steps should be completed without interruption and tests should be centrifuged
and read immediately after addition of anti-human globulin. Delays may result in dissociation
of antigen-antibody complexes, leading to false negative or weak positive reactions.
3. Caution should be exercised in the interpretation of results of tests performed at
temperatures other than those recommended.
LIMITATIONS
1. Red cells with a positive DAT due to a coating of IgG cannot be typed by the indirect
antiglobulin technique.
2. False positive results may occur due to the fact that agglutinins to albumin are found in a
small proportion of serum samples.
3. The efficacy of albumin reagent is to be controlled throughout their use.
4. Serological Albumin will no enhance the reactivity of all blood group antibodies.
5. Serological Albumin should not be used as negative controls for potentiated IgG blood
grouping reagents.
6. False positive or false negative results may occur due to:
Contamination of test materials
Improper storage, cell concentration, incubation time or temperature
Improper or excessive centrifugation
Introduction of human serum/gamma globulins into test
7. The user is responsible for the performance of the reagents by any methods other than
those mentioned in the Recommended Techniques.
8. Any deviations from the Recommended Techniques should be validated prior to use.
PERFORMANCE CHARACTERISTICS
1. The reagents have been characterised by the procedures mentioned in the
Recommended Techniques.
2. Prior to release, each lot of Spinreact 22% and 30% Serological Albumin have been
shown to enhance agglutination of Rh and other antibodies when used according to
Recommended Techniques.
3. Each lot is tested to assure specificity in an antibody-free system with red cells known to
possess the most frequently inherited blood group antigens.
4. The Quality Control of the reagents was performed using red cells that had been w ashed
with PBS prior to use.
5. The reagents comply with the recommendations contained in the latest issue of the
Guidelines for the UK Blood Transfusion Services.
